Methods of identifying obm modulators

ABSTRACT

Novel proteins and polypeptides binding to osteoclastogenesis inhibitory factor (OCIF) (OCIF-binding molecules, OBMs) and nucleic acids encoding these proteins and polypeptides are provided. Processes for producing these proteins, polypeptides, and nucleic acid molecules by genetic engineering are provided. Medicinal compounds are provided which comprise proteins and nucleic acids according to the invention, as well as proteins which bind to OBM, including anti-OBM antibodies. These compounds may be used for the treatment of bone disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/340,327, filed Dec. 19, 2008, which is a continuation of U.S.application Ser. No. 10/167,182, filed Jun. 11, 2002, now U.S. Pat. No.7,527,790, which is a divisional of U.S. application Ser. No.09/202,455, filed Dec. 15, 1998, now abandoned, which is a nationalphase under 35 U.S.C. §371 of International Application No.PCT/JP98/01728, filed Apr. 15, 1998, which claims priority to JapanesePatent Application No. 97808/1997, filed Apr. 15, 1997, Japanese PatentApplication No. 151434/1997, filed Jun. 9, 1997, Japanese PatentApplication No. 217897/1997, filed Aug. 12, 1997, Japanese PatentApplication No. 224803/1997, filed Aug. 21, 1997 and Japanese PatentApplication No. 332241/1997, filed Dec. 2, 1997. The contents of each ofthese related applications are hereby incorporated by reference in itsentirety.

FIELD OF TECHNOLOGY

The present invention relates to a novel protein (OCIF-binding molecule,the protein may be hereinafter called OBM) which binds toosteoclastogenesis inhibitory factor (hereinafter it may be called OCIF)and a method to produce this protein.

The present invention also relates to DNA encoding this protein,proteins containing the amino acid sequence encoded by this DNA, amethod for the preparation of this protein utilizing genetic engineeringtechniques, and pharmaceutical compositions comprising this protein.

The present invention further relates to methods for screening, usingthis protein and the DNA, substances to control the expression of thisprotein, substances inhibiting or regulating the biological activity ofthis protein, or receptors transducing the signal of the protein byinteracting with this protein, to substances obtained by the screening,and to pharmaceutical compositions which comprise the resultingsubstances.

The present invention further relates to antibodies against thisprotein, methods for preparing the antibodies, and pharmaceuticalcompositions comprising these antibodies.

BACKGROUND OF THE INVENTION

Bone metabolism is dependent on the overall activity of osteoblastswhich control bone formation and osteoclasts which control boneresorption. Abnormality of bone metabolism is considered to be caused byan imbalance of the bone formation and the bone resorption.Osteoporosis, hypercalcemia, Paget's disease, renal osteodystrophy,chronic rheumarthritis, osteoarthristis, and the like are known asdiseases accompanying abnormality of bone metabolism. Osteoporosis is atypical disease caused by such abnormality of bone metabolism. Thisdisease is generated when bone resorption by osteoclasts exceeds boneformation by osteoblasts. The disease is characterized by a decrease inboth the bone calcified material and the bone matrix. Although themechanism of this disease is not completely elucidated, the diseasecauses aches in bones, makes them fragile, and may result in fracturing.This disease is becoming a social problem because it increases thenumber of bedridden aged persons as the aged population becomes larger.Development of therapeutic agent for this disease is urgently desired.Disease due to a decrease in bone mass is expected to be cured bysuppressing bone resorption, accelerating bone formation, or improvingthe balance between bone resorption and formation. Bone formation isexpected to increase by accelerating proliferation, differentiation, oractivation of osteoblasts which form bone, or by suppressingproliferation, differentiation, or activation of osteoclasts whichresorb bone. In recent years, strong interest has been directed tohormones, low molecular weight substances, or physiologically activeproteins exhibiting such activities, and energetic basic research anddevelopment is underway on these subjects.

Drugs such as a calcitonin agents, active-form vitamin D₃ agents,hormone agents containing estradiol, ipriflavone, vitamin K₂, andbisphosphonate compounds have already been known as drugs to treat andshorten the treatment period of diseases related to bone. Clinical testsare in progress on active-form vitamin D₃ derivatives, estradiolderivatives, and bisphosphonate compounds of the second and the thirdgeneration to develop therapeutic agents with excellent efficacy andminimal side effects.

However, therapies using these agents were found not necessarilysatisfactory in terms of efficacy and therapeutic results. Developmentof novel therapeutic agents which are safer and with higher efficacy isurgently desired. Some agents used for the treatment of diseases relatedto bone metabolism are used only limitedly due to their side effects.Furthermore, treatments using two or more agents in combination arecurrently the mainstream in the treatment of diseases related to bonemetabolism such as osteoporosis. From such a point of view, developmentof drugs having action mechanisms different from those of conventionaldrugs, and exhibiting a higher efficacy and minimal side effects isdesired.

As mentioned above, the cells controlling bone metabolism areosteoblasts and osteoclasts. These cells are known to have close mutualinteractions called “coupling”. Specifically, cytokines such asInterleukins 1(IL-1), 3(IL-3), 6(IL-6), and 11(IL-11), granulocyticmacrophage-colony stimulating factor (GM-CSF), macrophage-colonystimulating factor (M-CSF), Interferon-γ (IFN-γ), tumor necrosis factorα (TNF-α), and transforming growth factor-β (TGF-β), secreted byosteoblastic stromal cells are known to accelerate or suppressdifferentiation or maturation of osteoclasts (Raisz: Disorders of Boneand Mineral Metabolism, 287-311, 1992; Suda et al.: Principles of BoneBiology, 87-102, 1996; Suda et al.: Endocrine Reviews, 4, 266-270, 1995,Lacey et al.: Endocrinology, 186, 2369-2376, 1995). It has been reportedthat osteoblastic stromal cells play an important role in thedifferentiation and maturation of osteoclasts, as well as in osteoclastfunctions such as bone resorption by mature osteoclasts, throughcell-to-cell contact with immature osteoclast precursors or matureosteoclasts.

A factor called osteoclast differentiation factor (ODF, Suda et al.:Endocrine Rev. 13, 66-80, 1992; Suda et al.: Bone 17, 87S-91S, 1995) isthought to be expressed on the membrane of osteoblastic stromal cellsand involved in the formation of osteoclasts through cell-to-cellcontact. According to this hypothesis, an ODF receptor is present in theprecursor cells of osteoclasts. However, so far neither the ODF nor thereceptor has been purified or identified. There are also no reportsrelating to their characteristics, action mechanism, or structure. Thus,the mechanism involved in differentiation and maturation of osteoclastshas not yet been sufficiently elucidated. Clarification of thismechanism will greatly contribute not only to the basic medicine, butalso to the development of novel drugs for the treatment of diseasesassociated with abnormality of bone metabolism.

The present inventors have conducted extensive studies in view of thissituation and discovered an osteoclastogenesis inhibitory factor (OCIF)in a culture broth of human embryonic lung fibroblast, IMR-90 (ATCCDeposition No. CCL186) (WO 96/26217).

The present inventors have been successful in cloning DNA encoding OCIF,production of recombinant OCIF in animal cells, and confirmation of invivo pharmaceutical effects (improving effect on bone metabolism, etc.)of the recombinant OCIF. OCIF is expected to be used as an agent for theprevention or treatment of diseases related to abnormality of bonemetabolism, with higher efficacy than conventional drugs and less sideeffects.

DISCLOSURE OF THE INVENTION

The present inventors have searched for a protein which binds toosteoclastogenesis inhibitory factor (OCIF) and discovered that anOCIF-binding protein is specifically expressed on the osteoblasticstromal cells cultured in the presence of a bone resorption factor suchas active-form vitamin D₃ and parathyroid hormone (PTH). In addition,the present inventors have investigated the characteristics andphysiological functions of this OCIF-binding protein and found that theprotein exhibits biological activity of a factor which supports orpromotes the osteoclast differentiation and maturation from immatureprecursors of osteoclasts. These findings have led to the completion ofthe present invention. Further investigation into the protein of thepresent invention has proven that this is an important proteincontrolling the differentiation and maturation of osteoclasts fromimmature precursors of osteoclasts in a co-culture system of theosteoblastic stromal cells and spleen cells. The success inidentification and isolation of the protein which functions as a factorsupporting or promoting differentiation and maturation of osteoclasts inthe present invention has enabled screening for a novel medicine usefulfor abnormality of bone metabolism based on mechanism of bone metabolismutilizing the protein of the present invention.

Accordingly, an object of the present invention is to provide a novelprotein (OCIF-binding molecule or OBM) which binds to osteoclastogenesisinhibitory factor (OCIF), and a method to produce this protein.

Another object of the present invention is to provide DNA encoding thisprotein, proteins containing an amino acid sequence encoded by this DNA,a method for producing this protein utilizing genetic engineeringtechniques, and pharmaceutical compositions comprising this protein.

A further object of the present invention is to provide methods forscreening substances which control expression of this protein using thisprotein and the DNA, substances inhibiting or regulating the biologicalactivity of this protein, receptors transducing the action of theprotein by binding to the protein, substances obtained by the screening,and pharmaceutical compositions which comprises these substances.

A still further object of the present invention is to provide antibodiesagainst this protein, methods for preparing the antibodies, andpharmaceutical compositions comprising these antibodies.

The protein of the present invention has the following physicochemicalproperties and biological activity.

(a) Affinity: specifically binds to the osteoclastogenesis inhibitoryfactor (OCIF) and exhibits high affinity to OCIF (dissociation constanton cell membrane: Kd=10⁻⁹ M or less);

(b) Molecular weight: has a molecular weight of approximately30,000-40,000 when determined by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) under non-reducing conditions and an apparent molecularweight of approximately 90,000-110,000 when cross-linked to a monomerform OCIF; and

(c) Biological activity: exhibits activity supporting or promotingosteoclast differentiation and maturation in a co-culture system of themouse osteoblastic stromal cells and mouse spleen cells in the presenceof bone resorption factors such as active-form vitamin D₃ andparathyroid hormone (PTH).

A co-culture system of ST2, a mouse osteoblastic stromal cell line, andmouse spleen cells in the presence of active-form vitamin D₃ or PTH iswell known as a typical in vitro culture system for osteoclastformation. The cells expressing the protein of the present invention canbe determined by testing the binding of OCIF to mouse osteoblasticstromal cells or mouse spleen cells cultured in the presence or absenceof active-form vitamin D₃. The protein of the present invention isspecified as the protein which is induced specifically on theosteoblastic stromal cells cultured in the presence of an osteotropicfactor such as active-form vitamin D₃ or PTH. In addition, the proteinof the invention can be specified as a protein exhibiting biologicalactivity supporting or promoting differentiation and maturation ofosteoclasts from the following results. That is, the osteoclastformation is inhibited dose dependently by the addition of 1 to 40 ng/mlof OCIF to the above-mentioned co-culture system in the presence of theactive-form vitamin D₃, the time course of expression of the protein ofthe present invention on ST2 cells in the presence of active-formvitamin D₃ well correlates with the time course of osteoclast formationin the co-culture. In addition, the amount of protein of the presentinvention expressed on ST2 cells correlates with the capability of thecells to support the osteoclast formation, and the binding of OCIF tothe protein of the present invention on the ST2 cells completelysuppresses osteoclasts formation.

The affinity of the protein of the present invention to OCIF can beevaluated by labeling OCIF and examining the binding of the labeled OCIFto the surface of animal cell membrane. OCIF can be labeled by aconventional protein-labeling method such as radioisotope or fluorescentlabeling. Labeling of tyrosine residues with ¹²⁵I can be given as aspecific example of labeling of the OCIF with an radioisotope. Labelingmethods such as iodogen method, chloramine T method, and enzymaticmethod can be utilized. The binding of the labeled OCIF to the surfacemembrane of animal cell can be tested by a conventional method. Theaddition of unlabeled OCIF to the medium used for the binding assay to aconcentration, 100 to 400 times the concentration of labeled OCIF,ensures measurement of non-specific binding. The amount of specificbinding of OCIF can be calculated by subtracting the amount ofnon-specific binding from the total amount of binding of the labeledOCIF. The affinity of the protein of the present invention expressed onthe cell membrane to OCIF can be evaluated by changing the amount oflabeled OCIF and analyzing the specific binding by Scatchard plot.

The determined affinity of the protein of the present invention to OCIFis approximately 100-500 pM. The protein of the present invention isspecified by a high affinity (dissociation constant on cell membrane:Kd=10⁻⁹ M or less) to osteoclastogenesis inhibitory factor (OCIF). Themolecular weight of OBM can be accessed by gel filtrationchromatography, SDS-PAGE, or the like. SDS-PAGE is preferred in order toaccurately determine the molecular weight. The OBM is specified as aprotein having a molecular weight of approximately 40,000 (40,000±4,000)under reducing conditions.

The protein of the present invention can be obtained from mouseosteoblastic stromal cell line, ST2, mouse preadipocyte cell line, PA6,human osteoblastic cell lines, or other osteoblastic cells selected frommammalians such as humans, mice, or rats. As the substances to induceexpression of the protein of the present invention, osteotropic factorssuch as active-form vitamin D₃ (calcitriol), parathyroid hormone (PTH),interleukin (IL)-1, IL-6, IL-11, Oncostatin M, and leukemia inhibitoryfactor (LIF) can be given. These substances can be added in theconcentration of 10⁻⁸ M (active-form vitamin D₃ and PTH), 10 ng/ml(IL-11), or 1 ng/ml (Oncostatin M). IL-6 is preferably used at aconcentration of 20 ng/ml with 500 ng/ml soluble IL-6 receptor.Preferably, confluent cells of mouse osteoblastic stromal cell line,ST2, cultured in α-MEM medium to which 10⁻⁸ M of active-form vitamin D₃,10⁻⁷ M of dexamethasone, and 10% fetal bovine serum were added can beused. The cultured cells may be collected by scraping with a cellscraper. The collected cells may be stored at −80° C. until use.

The protein of the present invention can be purified efficiently fromthe membrane fractions of the collected cells. The membrane fractionscan be prepared by a conventional method which is used to prepareintracellular organella. Various types of protease inhibitors may beadded to the buffer solution used for the preparation of the membranefractions. Examples of the protease inhibitors include serine proteaseinhibitors, thiol protease inhibitors, and metaprotease inhibitors.PMSF, APMSF, EDTA, o-phenanthroline, leupeptin, pepstatin A, aprotinin,and soybean trypsin inhibitor are givens as specific examples. A Dauncehomogenizer, a polytron homogenizer, or an ultrasonic processor can beused to homogenize the cells. The cell homogenate is suspended in abuffer solution containing 0.5 M of sucrose and centrifuged for 10minutes at 600×g, to separate the nucleus and undisrupted cells asprecipitate. The supernatant is centrifuged for 90 minutes at 150,000×gto obtain a membrane fraction as precipitate. The obtained membranefraction is treated by various types of detergents to efficientlysolubilize and extract the protein of the present invention from thecell membrane. Detergents which are commonly used to solubilize cellmembrane proteins, such as CHAPS(3-[(3-cholamidopropyl)-dimethylamo-nio]-1-propanesulfonate), TritonX-100, Nikkol, and n-octylglycoside, can be used. Preferably, 0.5% CHAPSis added to the membrane fraction and the mixture is stirred for 2 hoursat 4° C. to solubilize the protein of the present invention. The samplethus prepared is centrifuged at 150,000×g for 60 minutes to obtain thesolubilized membrane fraction as a supernatant.

The protein of the present invention can be purified from thesolubilized membrane fraction with a column, gel, or resin coupled withOCIF. The immobilized OCIF may be that isolated from a culture broth ofhuman embryonic lung fibroblasts, IMR-90, described in WO 96/26217 orrOCIF prepared using genetic engineering techniques. rOCIF can beprepared by introducing human cDNA, mouse cDNA, or rat cDNA into anexpression vector according to a conventional method, transducing theconstructed vector in animal cells such as CHO cells, BHK cells, orNamalwa cells, or in insect cells to produce rOCIF, and purifying rOCIF.Obtained OCIF has a molecular weight of approximately 60 kDa(monomer-form) or 120 kDa (dimer-form). The dimer-form OCIF ispreferable for immobilization. Given as examples of the gels and resinsto which OCIF is immobilized are ECH Sepharose 4B, EAH Sepharose 4B,Thiopropyl Sepharose 6B, CNBr-activated Sepharose 4B, activated CHSepharose 4B, Epoxy activated Sepharose 6B, activated thiol Sepharose 4B(these are manufactured by Pharmacia Co.), TSKgel AF-Epoxy Toyopal 650,TSKgel AF-Amino Toyopal 650, TSKgel AF-Formyl Toyopal 650, TSKgelAF-Carboxy Toyopal 650, TSKgel AF-Tresyl Toyopal 650 (these aremanufactured by Tosoh Corporation), Amino-Cellulofine,Carboxy-Cellulofine, FMP activated Cellulofine, Formyl-Cellulofine(these are manufactured by Seikagaku Kogyo Co.), Affigel 10, Affigel 15,and Affiprep 10 (these are manufactured by BioRad Co.). As columns towhich OCIF is immobilized, HiTrap NHS-activated column (Pharmacia Co.),TSKgel Tresyl-5PW (Tosoh Corporation), etc. can be given. As a specificexample of the method for immobilizing OCIF to a HiTrap NHS-activatedcolumn (1 ml, Pharmacia Co.), the following method can be given.Specifically, 1 ml of 0.2M NaHCO₃/0.5 M NaCl solution (pH 8.3)containing 13.0 mg of OCIF is injected to the column to perform couplingreaction at room temperature for 30 minutes. 0.5 M ethanolamine/0.5 MNaCl (pH 8.3) and 0.1 M acetic acid/0.5 M NaCl (pH 4.0) are sequentiallyapplied to the column. Then, the column is again washed with 0.5 Methanolamine/0.5 M NaCl (pH 8.3) and the column is allowed to stand forone hour at room temperature to block excess active groups. The columnis sequentially washed twice with 0.5 Methanolamine/0.5 M NaCl (pH 8.3)and 0.1 M acetic acid/0.5 M NaCl (pH 4.0), and then washed with 50 mMTris/1M NaCl/0.1% CHAPS solution (pH 7.5), thereby obtaining aOCIF-immobilized column. The protein of the present invention can beefficiently purified by an OCIF-immobilized column prepared in thismanner, or an OCIF-immobilized gel or resin.

It is desirable to add the various above-mentioned protease inhibitorsto the buffer solutions used for the purification of the protein tosuppress degradation of the protein of the present invention. Theprotein of the present invention can be purified by loading theabove-mentioned solubilized membrane fraction on the OCIF-immobilizedcolumn or by mixing with the OCIF-immobilized gel or resin, and elutingthe protein from the column, gel, or resin with acid, various proteindenaturing agents, cacodylate buffer, and the like. It is desirable touse an acid for elution and to neutralize immediately after elution tominimize denaturation of the protein of the present invention. As theacidic solution used for elution, 0.1 M glycine-hydrochloric acidsolution (pH 3.0), 0.1 M glycine-hydrochloric acid solution (pH 2.0),0.1 M sodium citrate solution (pH 2.0), and the like can be given.

The protein of the present invention can be further purified byconventional purification methods used for purification of variousproteins from biological materials and by various purification methodsutilizing the physicochemical properties of this protein. To concentratesolutions containing the protein of the present invention, conventionaltechniques used in the purification process for proteins such as ultrafiltration, freeze drying, and salting-out, can be used. Ultrafiltration with Centricon-10 (BioRad Co.), for example, is preferablyused. As a means for the purification, various techniques conventionallyutilized for the purification of proteins, such as ion exchangechromatography, gel filtration chromatography, hydrophobicchromatography, reverse phase chromatography, and preparativeelectrophoresis, are used in combination. More specifically, it ispossible to purify the protein of the present invention by a combinationof gel filtration chromatography with Superose 12 column (Pharmacia Co.)and reverse phase chromatography. To detect the protein of the presentinvention in the purification process, the binding activity of theprotein of the present invention to the immobilized OCIF is examined orthe material bound to the immobilized OCIF is immuno precipitated withan anti-OCIF antibody and analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE).

The obtained protein of the present invention is useful as an agent fortreating diseases caused by abnormality of bone metabolism such asosteopetrosis or as a reagent for research and diagnosis of thesediseases.

The present invention further provides DNA encoding a novel protein(OCIF-binding molecule or OBM) which binds to osteoclastogenesisinhibitory factor, proteins containing the amino acid sequence encodedby this DNA, a method for the preparation of this protein by the geneticengineering technique, and pharmaceutical compositions comprising thisprotein. Furthermore, the present invention provides methods forscreening substances to regulate expression of OBM, a method forscreening substances inhibiting or modifying the biological activity ofOBM, or a method for screening receptors transducing the action of OBMby binding to OBM, and pharmaceutical compositions which comprisessubstances obtained as a result of the screening.

The novel protein OBM which is encoded by the DNA of the presentinvention has the following physicochemical properties and biologicalactivity.

(a) binds specifically to osteoclastogenesis inhibitory factor (OCIF),

(b) has a molecular weight of approximately 40,000 (±4,000) whendetermined by SDS-PAGE under reducing conditions and an apparentmolecular weight of approximately 90,000-110,000 when crosslinked tomonomer-form OCIF, and

(c) exhibits activity supporting or promoting differentiation andmaturation of osteoclasts.

Human osteoclastogenesis inhibitory factor (OCIF) which is used as aprobe to identify the DNA encoding OBM, the OCIF-binding molecule of thepresent invention, and to evaluate properties of OBM can be isolatedfrom a culture broth of a human embryonic lung fibroblast cell line,IMR-90, according to WO No. 96/26217. Recombinant human OCIF,recombinant mouse OCIF, recombinant rat OCIF, and the like can also beused for the isolation and identification of the DNA coding OBM. Theserecombinant OCIF proteins can be produced by inserting DNA fragmentsencoding these proteins into expression vectors according toconventional methods, expressing in animal cells such as CHO cells, BHKcells, or Namalwa cells, or in insect cells, and purifying them.

As a method for isolating cDNA encoding a target protein (cDNA cloning),the method comprising determination of a partial amino acid sequence ofthe protein and isolation of the target cDNA by hybridization utilizingthe nucleotide sequence corresponding to the amino acid sequence can beemployed. Another available method, even in the case where the aminoacid sequence of the protein is not known, comprises constructing a cDNAlibrary in a expression vector, introducing the cDNA into cells, andscreening for the expression of the target protein to isolate theobjective cDNA (expression cloning method, D'Andrea et al.: Cell 57,277-285, 1989; Fukunaga et al.: Cell 61, 341-350, 1990). In theexpression cloning method, suitable host cells such as bacteria, yeast,animal cells, and the like are selected depending on the objective. Inmany cases, animal cells are selected as the host cells for cloning cDNAencoding a protein such as the protein of the present invention which isconsidered to be present in animal cell membrane surface. Normally, hostcells showing high efficiency for DNA transfection and achievingexpression of the introduced DNA at high levels are selected. One ofsuch animal cells is the monkey kidney cells, COS-7, used in the presentinvention. Because SV40 large T antigen is expressed in the COS-7 cell,a plasmid which has a replicator of SV40 can be present as an episome ofmultiple copies in the cell, so that a high level of expression isexpected. In addition, because expression of a target protein by COS-7cells reaches a maximum within a few days after introduction of DNA, thecell is suitable for rapid screening. A combination of this host cellwith a plasmid capable of high expression ensures gene expression of anextremely high level. The factor exhibiting the greatest influence onthe expression of a gene on a plasmid is a promoter. Promoters such asSRα promoter and cytomegalovirus-derived promoters are used as highexpression promoters. To screen for the cDNA encoding a membrane proteinby the expression cloning strategy, screening procedures such as bindingmethod, panning method, or film emulsion method are used.

The present invention relates to DNA encoding the protein (OBM) whichspecifically binds to OCIF, isolated by the combination of theexpression cloning strategy and the screening by the binding method, tothe expressed protein, and to screening of physiologically activesubstances using the DNA or the expressed protein. OBM encoded by theDNA of the present invention can be detected by labeling OCIF andtesting the binding of the labeled OCIF to membrane surface of an animalcell. OCIF can be labeled by a conventional labeling method such asradioisotope labeling method or fluorescent labeling method which isused for labeling common proteins. Labeling tyrosine residues by ¹²⁵Ican be given as a specific example of labeling OCIF with a radioisotope.Labeling methods such as the iodogene method, chloramine T method, andenzymatic method can be utilized. The binding of labeled OCIF to theanimal cell membrane surface can be tested by conventional methods. Theaddition of unlabeled OCIF to the medium used for the test to aconcentration, 100 to 400 times the concentration of labeled OCIF,enables quantification of the amount of non-specific binding. The amountof specific binding of OCIF can be calculated by subtracting the amountof non-specific binding from the total amount of binding of the labeledOCIF.

The present inventors assumed that there is interaction between thefactor involved in differentiation of osteoclasts and OCIF. Based onthis assumption, to isolate the protein to which recombinant OCIF binds,the inventors screened the expression library prepared from mRNA ofmouse osteoblastic stromal cell line, ST2, according to the followingmethod. Specifically, DNA synthesized using ST2 mRNA was inserted intoan expression vector for animal cells and the vector with the insert wasintroduced into monkey kidney COS-7 cells. The objective proteinexpressed on the COS-7 cells was screened using OCIF labeled with ¹²⁵Ias a probe. As a result, DNA encoding the protein which bindsspecifically to OCIF was isolated. The nucleotide sequence of the DNAencoding this OCIF-binding molecule (OCIF-binding molecule; OBM) wasthen determined. Moreover, OBM encoded by this DNA was found to bindspecifically and strongly to OCIF, on the cell membrane.

Comparatively mild conditions for hybridization of DNA in the presentinvention are the conditions, for example, wherein DNA is transferred toa nylon membrane and immobilized thereto according to conventionalmethods and hybridized in a buffer solution for hybridization with aprobe DNA labeled with an isotope at a temperature of 40-70° C. forabout 2 hours to overnight, followed by washing in 0.5×SSC (0.075 Msodium chloride and 0.0075 M sodium citrate) at 45° C. for 10 minutes.Specifically, Highbond N (Amersham Co.) is used as the nylon membrane totransfer and immobilize DNA thereon. DNA is then hybridized with a probeDNA labeled with ³²P in a rapid hybridization buffer (Amersham Co.) at65° C. for 2 hours, followed by washing with 0.5×SSC (0.075 M sodiumchloride and 0.0075 M sodium citrate) at 45° C. for 10 minutes.

A co-culture system of mouse osteoblastic stromal cells, ST2, and mousespleen cells in the presence of active-form vitamin D₃ or PTH is wellknown as a typical in vitro culture system for osteoclast-formation. Theprotein of the present invention is specified as the protein which isinduced specifically on the osteoblastic stromal cells cultured in thepresence of an agent which accelerates bone resorption such asactive-form vitamin D₃ or PTH. In addition, because of the fact thatformation of osteoclasts is stimulated by the addition of the proteinencoded by the DNA of the present invention to mouse spleen cellscultured even in the absence of active-form vitamin D₃ or PTH, OBM whichis encoded by the DNA of the present invention is considered to beinvolved in the differentiation and maturation of osteoclasts.

Recombinant OBM can be produced by inserting the DNA of the presentinvention into an expression vector to construct a plasmid andintroducing the plasmid into various cells or microorganisms to expressrecombinant OBM. As a host in which recombinant OBM is expressed,mammalian cells such as COS-7, CHO, Namalwa, or bacteria such asEscherichia coli can be used. OBM may be expressed as amembrane-bound-form protein using the full length DNA or as asecretion-form or a soluble-form protein by removing the portionencoding the transmembrane domain. The produced recombinant OBM can beefficiently purified using a suitable combination of conventionalpurification methods used for common proteins, such as affinitychromatography using OCIF-immobilized columns, ion exchangechromatography, and gel filtration chromatography. The obtained proteinof the present invention is useful as an agent for treating diseasescaused by abnormality of bone metabolism such as osteopetrosis or as areagent for research and diagnosis of such diseases.

The following screening operations can be carried out using the proteinOBM encoded by the DNA of the present invention: (1) screening ofsubstances which regulate expression of OBM, (2) screening of substanceswhich specifically bind to OBM and inhibit the biological activity ofOBM, and (3) screening of proteins which are present in osteoclastprecursor cells and transduce the biological activity of OBM (OBMreceptor). It is also possible to develop antagonists and agonists usingthis OBM receptor. In the combinatorial chemistry using theabove-mentioned OBM or OBM receptor, a peptide library used for thescreening of the antagonists or agonists can be prepared by thefollowing method. Specifically, one of the methods is split method (Lamet al.; Nature 354, 82-84, 1991). According to this method, syntheticcarriers (beads) each comprising a specific amino acid (unit) boundthereto are prepared separately for all units. The synthesized carriersare mixed altogether and divided into portions equal to the number ofthe units. Then, the next units are bound. This procedure is repeated“n” times to produce a library containing carriers to which “n” unitsare bound. According to this synthetic method, each carrier pool has onetype of sequence. Therefore, it is possible to identify a peptidespecifically binding to the protein of the present invention byselecting the pool which gives a signal positive in this screeningmethod using the protein of the present invention, and determining theamino acid sequence of the peptide bound on the pool. Another method isphage display method which utilizes phages carrying synthetic DNA whichencode peptides with random amino acid sequences. The method has theadvantage of increasing the number of molecules in the library ascompared with the above-mentioned synthetic peptide library method, buthas the disadvantage of less variety for a given number of moleculesbecause there can be particular sequences which are missing in thelibrary if the phages are unable to express those sequences. In thephage display method, the screening system using the protein of thepresent invention can also be applied to determine the nucleotidesequence encoding the peptide. That is, the phage specifically bindingto the protein of the present invention is concentrated by panning, theselected phage is amplified in Escherichia coli, and the nucleotidesequence encoding the peptide is determined. In addition, a peptideexhibiting high specificity and high affinity to OBM or OBM receptor canbe screened from a peptide library using the screening systems mentionedabove in (2) and (3) by screening in the presence of OBM or OCIF whileincreasing the concentration of OBM or OCIF. Only positive carrier poolsor phages are selected in this manner. For example, low molecular weightpeptide agonists exhibiting an EPO (erythropoietin)-like activity werescreened from a peptide library using a receptor of erythropoietin (EPO)which is a hematopoietic hormone, the tertiary structure of thissubstance was analyzed, and based on this tertiary structure, lowmolecular weight substances (antagonist) exhibiting the EPO-likeactivity were synthesized (Nicholas et al.: Science, 273, 458-463,1996).

The present inventors have previously discovered using theosteoclastogenesis inhibitory factor, OCIF, that an OCIF-binding proteinis specifically expressed on osteoblastic stromal cell line, ST2,cultured in the presence of a osteotropic factor such as active-formvitamin D₃ or parathyroid hormone (PTH). The inventors further foundthat this protein exhibits a biological activity to support or stimulatedifferentiation or maturation of osteoclasts from immature osteoclastprecursor cells, and clarified various physicochemical properties andthe biological activity of this protein by purification thereof. Inorder to compare the recombinant OBM expressed by the DNA of the presentinvention and the above-mentioned purified natural type protein whichspecifically binds to OCIF, the present inventors investigated thephysicochemical properties and biological activities of the twoproteins. As a result, the two proteins were confirmed (1) to be bothmembrane-bound proteins which specifically bind to OCIF, (2) to havemolecular weights of approximately 40,000 determined by SDS-PAGE, and(3) to have apparent molecular weights of about 90,000-110,000 whencross-linked to a monomer form OCIF. Not only are these physicochemicalproperties identical, but both proteins exhibit a biological activity tosupport or stimulate differentiation or maturation of osteoclasts,suggesting the possibility that these are the same protein. In addition,a rabbit anti-OBM polyclonal antibody produced using the purifiedprotein prepared by expressing the DNA of the present invention by agenetic engineering technique (recombinant OBM) was confirmed to crossreact with the above-described purified natural type protein, to inhibitspecific binding of this purified natural type protein and OCIF in thesame manner as the antibody inhibits specific binding of OBM and OCIF.Based on these results, it is clear that the recombinant OBM expressedby the DNA of the present invention is identical to the natural typeprotein which specifically binds to OCIF.

To isolate a gene (cDNA) encoding human OCIF-binding protein(hereinafter called human OBM) which specifically binds to OCIF andexhibits the activity to support and stimulate differentiation andmaturation of osteoclasts from mouse spleen cells in the same manner asthe natural type or recombinant mouse OBM dose, a cDNA library preparedfrom mRNA derived from human lymph nodes was screened using a human OBMcDNA fragment as a probe. The human OBM cDNA fragment was obtained bypolymerase chain reaction (PCR) in accordance with the method mentionedabove using both cDNA prepared from human lymph node as a template andthe primer which was prepared from mouse OBM cDNA. As a result, cDNAencoding the human protein which specifically binds to OCIF was isolatedand the nucleotide sequence of the cDNA encoding this human OCIF-bindingprotein molecule (i.e. the cDNA encoding human OBM) was determined.Similar to mouse OBM, this human OBM encoded by the cDNA hascharacteristics to bind to OCIF strongly and specifically on the cellmembrane and exhibits the activity to support and promotedifferentiation and maturation of osteoclasts from mouse spleen cells.Specifically, the present invention provides DNA encoding novel humanOBM protein which binds to osteoclastogenesis inhibitory factor (OCIF),a protein which possesses the amino acid sequence encoded by the DNA, amethod for producing the protein exhibiting characteristics ofspecifically binding to OCIF and the activity to support and promotedifferentiation and maturation of osteoclasts from mouse spleen cells bygenetic engineering techniques, pharmaceutical compositions comprisingthis protein for the treatment of diseases caused by abnormality of bonemetabolism, a method for screening substances regulating expression ofhuman OBM, a method for screening substances which inhibit or modulatethe activity of human OBM by binding to it, a method for screeningreceptors which bind to human OBM and transmit the action of OBM, and apharmaceutical compositions comprising the substances obtained by thesescreenings.

The present invention further provides DNA encoding novel human OBMprotein which specifically binds to OCIF and exhibits the biologicalactivity to support and promote differentiation and maturation ofosteoclasts, a protein which possesses the amino acid sequence encodedby the DNA, a method for producing the protein exhibitingcharacteristics of specifically binding to OCIF and the activity tosupport and promote differentiation and maturation of osteoclasts bygenetic engineering techniques, and pharmaceutical compositionscomprising this protein for the treatment of diseases causingabnormality of bone metabolism. Furthermore, the present inventionprovides a method for screening substances regulating expression ofhuman OBM, a method for screening substances which inhibit or modulatethe activity of human OBM by binding to it, a method for screeningreceptors binding to human OBM and transmitting the action of OBM,antibodies against human OCIF binding protein, and pharmaceuticalcompositions comprising these antibodies for the prevention or treatmentof diseases causing abnormality of bone metabolism.

The novel, human OCIF-binding protein molecule (OBM) which is encoded bythe DNA of the present invention has the following physicochemicalproperties and biological activity.

(a) binds specifically to osteoclastogenesis inhibitory factor (OCIF)(WO 96/26217),

(b) has a molecular weight of approximately 40,000 (±5,000) whendetermined by SDS-PAGE under reducing conditions and an apparentmolecular weight of approximately 90,000-110,000 when crosslinked with amonomer form OCIF, and

(c) exhibits activity to support and stimulate differentiation andmaturation of osteoclasts.

Mouse OBM cDNA which encodes mouse OCIF-binding protein and used as aprobe to isolate and identify the cDNA encoding human OBM of the presentinvention, can be isolated according to the above-mentioned method froma cDNA library of mouse osteoblastic stromal cell line, ST2. Humanosteoclastogenesis inhibitory factor (OCIF) which is necessary toevaluate the properties and the biological activity of the proteinobtained by expression of human OBM cDNA, can be prepared according tothe method described in WO 96/26217 by isolating from a culture broth ofhuman fibroblast cell line, IMR-90, or by genetic engineering techniquesusing the DNA encoding OCIF. Recombinant human OCIF, recombinant mouseOCIF, recombinant rat OCIF, or the like can be used for the assessmentof the properties and biological activity of human OBM. Theserecombinant OCIF can be obtained according to conventional methods byinserting cDNA into an expression vector, expressing the cDNA in animalcells such as CHO cells, BHK cells, or Namalwa cells, or in insectcells, and purifying the expressed proteins.

The following methods can be used to isolate human cDNA encoding thetarget protein (cDNA cloning). (1) A method comprising purifying theprotein, determining the partial amino acid sequence of the protein, andisolating the target cDNA by hybridization using the DNA fragmentcomprising nucleotide sequence corresponding to the amino acid sequenceas a probe, (2) a method applied even in the case where the amino acidsequence of the protein is not known, which comprises constructing acDNA library in a expression vector, introducing the cDNA library intocells, and screening for the expression of the target protein to isolatethe objective cDNA (expression cloning method), and (3) a method ofisolating cDNA encoding the target human protein from the cDNA libraryconstructed using human cells or tissues by hybridization or by the useof polymerase chain reaction (PCR) using the cDNA encoding the proteinof mammalian origin (other than human) which possesses the samecharacteristics and biological activity as the target protein of humanorigin as a probe, assuming that the cDNA probe has high homology withthe human-origin cDNA which to be cloned. Based on the assumption thathuman OBM cDNA has a high homology with mouse OBM cDNA, it is possibleto determine which cells or tissues produce human OBM by Northernhybridization method using the mouse OBM cDNA as a probe. Human OBM cDNAcan be obtained by the following method using the mouse OBM primerprepared from the mouse OBM cDNA. Human OBM cDNA fragments can beprepared by the PCR method using cDNA prepared from human OBM-producingtissues such as human lymph nodes as a template. These human OBM cDNAfragments are used as probes for screening the cDNA library of humanOBM-producing cells or tissues which were identified according to themethod mentioned above. The present invention relates to the DNAencoding human OBM which has characteristics of specific binding to OCIFand exhibits activity to support and promote differentiation andmaturation of osteoclasts. Because the OBM which is encoded by the DNAof the present invention is a membrane-bound type protein whichcomprises a transmembrane domain, this protein can be detected bylabeling OCIF and by examining the binding of the labeled OCIF to thesurface of animal cells in which the cDNA of the present invention wasexpressed. The above-described labeling method using radioisotope orfluoresceine conventionally applied to labeling proteins can be used forlabeling OCIF.

The molecular weight of the protein expressed by the human OBM cDNA ofthe present invention can be accessed by gel filtration chromatography,SDS-PAGE, or the like. In order to accurately determine the molecularweight, it is desirable to use the SDS-PAGE method, by which human OBMwas specified as a protein having a molecular weight of approximately40,000 (40,000±5,000) under reducing conditions.

Comparatively mild conditions for hybridization of DNA in the presentinvention are the conditions, for example, wherein DNA is transferred toa nylon membrane and immobilized thereto according to a conventionalmethod and hybridized with a probe DNA labeled with an isotope in abuffer solution for hybridization at a temperature of 40-70° C. forabout 2 hours to overnight, followed by washing in 0.5×SSC (0.075 Msodium chloride and 0.0075 M sodium citrate) at 45° C. for 10 minutes.Specifically, Highbond N (Amersham Co.) is used as the nylon membrane totransfer and immobilize DNA thereon. The DNA is then hybridized with aprobe DNA labeled with ³²P in a rapid hybridization buffer (AmershamCo.) at 65° C. for 2 hours, followed by washing with 0.5×SSC at 45° C.for 10 minutes.

A co-culture system of mouse osteoblastic stromal cells, ST2, and mousespleen cells in the presence of active-form vitamin D₃ or PTH is wellknown as a typical in vitro culture system for osteoclast-formation.Interaction by adhesion of osteoblastic stromal cells and spleen cellsand presence of an osteotropic factor such as active-form vitamin D₃ orPTH are indispensable for the osteoclasts formation in this in vitroculture system. In this in vitro culture system, COS cells, monkeykidney cells having no osteoclast formation-supporting capability,acquire capability to support osteoclasts formation from spleen cells inthe absence of an osteotropic factor when the cDNA of the presentinvention was expressed as osteoblastic stromal cell line ST2 did. Basedon the fact that the cDNA of the present invention encodes a proteincomprising a transmembrane domain form, this cDNA can be expressed as asecretion form or soluble-form by removing the part which encodes thistransmembrane domain. It was confirmed that osteoclasts can be formed bythe addition of the secretion form human OBM to the above-mentioned invitro culture system in the absence of osteotropic factors. Based onthese results, the human OBM which is encoded by the cDNA of the presentinvention is specified as the factor involved in the differentiation andmaturation of osteoclasts.

A recombinant human OBM can be prepared by inserting the cDNA of thepresent invention into an expression vector, preparing a human OBMexpression plasmid, introducing the plasmid into various cell strainsand expressing OBM in the cells. Mammalian cells such as COS-7, CHO,Namalwa cells, or bacteria such as Escherichia coli can be used as ahost for expressing OBM. In this case, OBM may be expressed as amembrane-bound-form protein, using the full length DNA, or as asecretion-form or soluble-form protein by removing the part encoding thetransmembrane domain. The recombinant OBM thus produced can beefficiently purified using a suitable combination of conventionalpurification methods used for common proteins such as affinitychromatography using OCIF immobilized columns, ion exchangechromatography, and gel filtration chromatography. Human OBM of thepresent invention thus obtained is useful as an agent for treatingdiseases caused by abnormality of bone metabolism such as osteopetrosisor as a reagent for research and diagnosis of such diseases.

The following screening operations can be carried out using the proteinOBM encoded by the DNA of the present invention: (1) screening ofsubstances which can regulate expression of human OBM, (2) screening ofsubstances which specifically bind to human OBM and inhibit or modifythe biological activity of OBM, and (3) screening of human proteinswhich are present in osteoclast precursor cells and transmit thebiological activity of human OBM (human OBM receptor). It is alsopossible to develop antagonists and agonists using this human OBMreceptor. In the combinatorial chemistry using the human OBM or humanOBM receptor, peptide libraries required for the screening ofantagonists or agonists can be produced by the same method as used forthe screening using mouse OBM. A peptide with extremely high specificityand affinity can be obtained by screening peptide libraries using humanOBM instead of mouse OBM.

Although this OBM is very useful as mentioned above and antibodiesspecifically recognizing OBM and enzyme immunoassay using theseantibodies are indispensable in determination of OBM concentration, noantibodies useful for the access of OBM concentration have been so faravailable. In addition, an anti-OBM antibody or anti-sOBM antibody whichneutralizes the biological activity of OBM or sOBM is supposed tosuppress the activity of OBM or sOBM, specifically the activity toinduce osteoclasts formation. These are expected to be useful astherapeutic agents to treat abnormality of bone metabolism. However, nosuch antibodies have so far been available.

In view of this situation, the present inventors have conductedextensive studies. As a result, the present inventors have foundantibodies (anti-OBM/sOBM antibodies) which recognize both OBM, amembrane-bound protein which specifically binds to osteoclastogenesisinhibitory factor (OCIF), and soluble OBM (sOBM) which lack atransmembrane domain. Accordingly, the present invention providesantibodies (anti-OBM/sOBM antibodies) which recognizes both OBM, amembrane-bound protein which specifically binds to osteoclastogenesisinhibitory factor (OCIF), and sOBM which lack a transmembrane domain; amethod for the preparation thereof; a method for determination of OBMand sOBM concentrations using these antibodies; and agents for theprevention or treatment of diseases resulting from abnormality of bonemetabolism.

The present invention relates to antibodies (anti-OBM/sOBM antibodies)which recognize both the OBM, a membrane-bound protein whichspecifically binds to osteoclastogenesis inhibitory factor (OCIF), andsoluble OBM (sOBM) which lack a transmembrane domain; a method for thepreparation thereof; a method for quantifying OBM and sOBM using theseantibodies; and agents for the prevention or treatment of diseasesresulting from abnormality of bone metabolism.

The antibodies of the present invention exhibit activity of neutralizingthe osteoclastogenesis accelerating activity which is the biologicalactivity of OBM and sOBM and comprises the antibodies having thefollowing characteristics:

(a) polyclonal antibody which recognizes both mouse OBM and mouse sOBM(anti-mouse OBM/sOBM polyclonal antibody),

(b) polyclonal antibody which recognizes both human OBM and human sOBM(anti-human OBM/sOBM polyclonal antibody),

(c) monoclonal antibodies which recognizes both mouse OBM and mouse sOBM(anti-mouse OBM/sOBM monoclonal antibodies),

(d) monoclonal antibodies which recognize both human OBM and human sOBM(anti-human OBM/sOBM monoclonal antibodies), and

(e) anti-human OBM/sOBM monoclonal antibodies which crossreact to bothmouse OBM and mouse sOBM.

The polyclonal antibody which recognizes both mouse OBM and mouse sOBM(hereinafter called anti-mouse OBM/sOBM polyclonal antibody) and thepolyclonal antibody which recognizes both human OBM and human sOBM(hereinafter called anti-human OBM/sOBM polyclonal antibody) wereproduced by the following method. The purified mouse OBM used as anantigen for immunization can be obtained according to theabove-mentioned method. Especially, mouse osteoblastic stromal cellline, ST2, was treated with active-form vitamin D₃, and OBM on the cellmembrane was purified using an OCIF-immobilized column and gelfiltration chromatography, thereby obtaining natural mouse OBM (nativeOBM). The above-mentioned mouse OBM cDNA (SEQ ID NO:15) or human OBMcDNA (SEQ ID NO:12) was inserted into an expression vector according toconventional methods. Recombinant mouse OBM (SEQ ID NO:1) andrecombinant human OBM (SEQ ID NO:11) can be obtained by expressing cDNAin animal cells such as CHO cells, BHK cells, Namalwa, or COS-7 cells,insect cells or Escherichia coli, and purifying them using the samepurification methods as mentioned above. These may be used as antigensfor immunization. In this instance, purifying a large amount and a highlevel of mouse OBM or human OBM which are membrane-bound proteins is atask requiring a great deal of labor. On the other hand, as mentionedabove, OBM which is a membrane-bound protein and a soluble OBM (sOBM)which is obtained by deleting transmembrane domain of OBM are known tobe almost the same in their osteoclast differentiation and maturationactivities. It is possible to use mouse sOBM and human sOBM which arerelatively easily expressed and purified to a high level, as antigensfor immunization.

Mouse sOBM (SEQ ID NO:16) and human sOBM (SEQ ID NO:17) can be obtainedby adding a nucleotide sequence encoding a known signal sequenceoriginating from the other secretion protein in the upstream side of the5′ end of, respectively, mouse sOBM cDNA (SEQ ID NO:18) and human sOBMcDNA (SEQ ID NO:19), inserting these into an expression vector by theuse of genetic engineering techniques, causing these proteins to beexpressed in host cells such as various animal cells, insect cells, orEscherichia coli, and purifying the resultant products. The antigens forimmunization thus obtained are dissolved in phosphate buffered saline(PBS), mixed with the same volume of Freund's complete adjuvant toemulsify the solution if required, and subcutaneously administered toanimals about once a week to immunize these animals several times. Abooster injection is given when the antibody titer reaches a maximum.Exsanguination is performed 10 days after the booster administration.The resulting antiserum is treated with ammonium sulfate precipitation.IgG fraction is purified using an anion exchange chromatography orpurified by protein A- or protein G-Sepharose column chromatographyafter diluting the antiserum two-fold with Binding Buffer™ (BioRad Co.),to obtain the anti-mouse or anti-human OBM/sOBM polyclonal antibody.

The monoclonal antibodies of the present invention can be obtainedaccording to the following method. In the same manner as in the case ofthe polyclonal antibodies, natural mouse OBM (native OBM), recombinantmouse or human OBM, or recombinant mouse or human sOBM can be used asimmunogens to prepare monoclonal antibodies. Hybridomas are producedaccording to conventional methods by immunizing mammals with theseantigens or by immunizing lymphocytes in vitro and fusing the immunizedcells with myeloma cells. By analyzing the hybridoma culture supernatantthus obtained by a solid phase ELISA method, antibody-producinghybridomas recognizing the highly purified antigen are selected. Theresulting hybridomas are cloned and established as stableantibody-producing hybridoma clones. These hybridomas are cultured toobtain the antibodies. Small mammals such as mice or rats are commonlyused to produce hybridomas. Animals are immunized by intravenously orintraperitoneally injecting the antigen diluted to a suitableconcentration using a suitable solvent such as physiological saltsolution. Optionally, Freund's complete adjuvant maybe used togetherwith antigen. These are usually injected 3-4 times, once a week or everytwo weeks. The immunized animals are dissected three days after finalimmunization. Splenocytes from the removed spleen are used as immunizedcells. As mouse myeloma to be fused with immunized cells, p3/x63-Ag8,p3-U1, NS-1, MPC-11, SP-2/0, FO, P3x63 Ag8.653, and 5194 can be given. Acell line such as R-210 is given as the cell of rat origin. Humanantibodies are produced by immunizing human B lymphocytes in vitro andfusing the immunized cells with human myeloma cells or a cell linetransformed with EB virus. The fusion of the immunized cells and myelomacells can be carried out according to a conventional method such as themethod of Koehler and Milstein (Koehler et al.: Nature 256, 495-497(1975)). A method using electric pulse is also applicable. Immunizedlymphocytes and myeloma cells are mixed at a conventionally acceptedratio and fused in an FCS-free (fetal bovine serum-free) culture mediumwith an addition of polyethylene glycol, and cultured in anFCS-containing HAT selection medium to select fused cells (hybridomas).Next, the hybridomas which produce antibodies were selected by using aconventional antibody detection method such as an ELISA, a plaquetechnique, Ouchterlony method, or aggregation method, to establishstable hybridomas. The hybridomas established in this way can besubcultured by a conventional culture method or can be stored byfreezing as required. A hybridoma can be cultured by a conventionalmethod to collect the culture supernatant or implanted in the abdominalcavity of mammals to obtain the antibody from the ascitic fluid. Theantibody in the culture supernatant or ascitic fluid can be purified bya conventional method such as salting out, ion exchange and gelfiltration chromatography, or protein A or protein G affinitychromatography. Almost all monoclonal antibodies obtained using sOBM asan antigen can specifically recognize not only sOBM but also OBM (suchantibodies are hereinafter called anti-OBM/sOBM monoclonal antibodies).These antibodies can be used for the quantification of OBM or sOBM. Theamounts of OBM and sOBM can be quantified by labeling these antibodieswith a radioisotope or an enzyme and by applying the labeled antibodiesto a quantification system known as a radioimmunoassay (RIA) or enzymeimmunoassay (EIA). Using these quantification systems, the amount ofsOBM in a biological sample such as blood or urine can be determinedwith ease at high sensitivity. In addition, the amount of OBM binding toa tissue or surface of cells can be measured with ease at highsensitivity utilizing a binding assay using these antibodies.

When an antibody is used as a medication for humans, it is desirable touse a human-type anti-human OBM/sOBM antibody in view of antigenicity.The human-type anti-human OBM/sOBM antibody can be prepared according tothe following methods (1), (2), or over (3). In the method (1), humanlymphocytes collected from human peripheral blood or spleen areimmunized with an antigen human OBM or human sOBM in vitro in thepresence of IL-4. The resulting immunized human lymphocytes are fusedwith K6H6/B5 (ATCC CRL1823) which is a hetero hybridoma of mouse andhuman, and screened to obtain the objective antibody producinghybridoma. The antibodies produced by the resulting antibody producinghybridomas are human type anti-human OBM/sOBM monoclonal antibodies. Theantibodies neutralizing the activity of human OBM/sOBM are selected fromthese antibodies. However, in general, it is difficult to produce anantibody exhibiting high affinity to an antigen by the method ofimmunizing human lymphocytes in vitro. Therefore, in order to obtainmonoclonal antibodies with high affinity to human OBM and sOBM, it isnecessary to increase the affinity of the human-type anti-human OBM/sOBMmonoclonal antibodies obtained by the above method. This can be doneaccording to the following method. First, a random mutation isintroduced into CDR region (particularly CDR3 region) of a human-typeanti-human OBM/sOBM monoclonal antibody which neutralizes OBM but has alow affinity, and makes the phage to express protein. Phages which canstrongly bind to human OBM/sOBM which are selected by a phage displaymethod using plates on which human OBM/sOBM antigens are immobilized.The selected phages are grown in Escherichia coli. The amino acidsequence of the CDR which exhibits high affinity is determined from thenucleotide sequence of the DNA cloned in the phage. The thus-obtainedDNA encoding the human type anti-human OBM/sOBM monoclonal antibodies isintroduced into a commonly used expression vector for mammalian cells toproduce the human type anti-human OBM/sOBM monoclonal antibodies. Thetarget human type anti-human OBM/sOBM monoclonal antibodies exhibitinghigh affinity and capable of neutralizing the biological activity ofhuman OBM/sOBM can be selected from these monoclonal antibodies. In themethod (2), mouse type anti-human OBM/sOBM monoclonal antibodies areproduced according to the same method as in the present invention usingBALB/cmouse (Koehler et al.: Nature 256, 495-49, 1975), and monoclonalantibodies which can neutralize the biological activity of humanOBM/sOBM and exhibiting high affinity are selected. These high affinitymouse anti-human OBM/sOBM monoclonal antibodies can be converted intohuman-type using the CDR-grafting technique (Winter and Milstein: Nature349, 293-299, 1991) by implanting its CDR regions (CDR-1, 2 and 3) intothe CDR regions of human IgG. In the method (3), human peripheral bloodlymphocytes are implanted into a severe combined immune deficiency(SCID) mouse. Because the implanted SCID mouse can produce humanantibodies (Mosier D. E. et al.: Nature 335, 256-259, 1988; Duchosal M.A. et al.: Nature 355, 258-262, 1992), lymphocytes which can produce thehuman monoclonal antibodies having specificity to human OBM/sOBM can becollected by screening SCID mouse immunized with human OBM or sOBM. Theresulting lymphocytes are fused with K6H6/B5 (ATCC CRL1823) which is aheterohybridoma of mouse and human, according to the procedure describedabove for the human antibodies in the method (1). The resultinghybridomas are screened to obtain hybridomas which can produce theobjective human monoclonal antibodies. The thus-obtained hybridomas arecultured to produce large amounts of the objective human monoclonalantibodies. The antibodies can be purified by the above-mentionedpurification method. In addition, it is possible to produce recombinanthuman monoclonal antibodies in large amounts by constructing a cDNAlibrary from the hybridoma which can produce the objective humanmonoclonal antibodies to obtain a gene (cDNA) encoding the objectivehuman-type monoclonal antibodies by cloning, inserting this gene into asuitable expression vector by using genetic engineering techniques, andexpressing the monoclonal antibodies in host cells such as variousanimal cells, insect cells, or Escherichia coli. A large amount ofpurified human monoclonal antibodies can be obtained by purifying fromthe resulting culture supernatant by the purification methods mentionedabove.

The antibodies which can neutralize the biological activity of OBM/sOBMcan be obtained from the anti-OBM/sOBM monoclonal antibodies producedaccording to this method. The antibodies which neutralize the biologicalactivity of OBM/sOBM are expected to be useful as agents for thetreatment or prevention of bone metabolism abnormality because of theircapability of blocking in vivo biological activity of OBM/sOBM,specifically the capability of preventing the induction osteoclastformation. The activity of anti-OBM/sOBM antibodies to neutralize thebiological activity of OBM or sOBM can be measured by determining theactivity to suppress osteoclast formation in the in vitro system.Specifically, the following in vitro osteoclastogenesis culture systemcan be given: (1) a co-culture system of mouse osteoblastic stromal cellstrain, ST2 cells, and mouse spleen cells in the presence of active-formvitamin D₃ and dexamethasone, (2) a co-culture system comprising OBMexpressing monkey kidney cell strain, COS-7, immobilizing theOBM-expressing cells with formaldehyde, and culturing mouse spleen cellson those cells in the presence of M-CSF, and (3) a culture system ofmouse spleen cells in the presence of recombinant sOBM and M-CSF. Theosteoclastogenesis-inhibitory activity of the anti-OBM/sOBM antibodiescan be measured by adding the anti-OBM/sOBM antibodies at variousconcentrations to these culture systems and investigating their effectson osteoclast formation. The osteoclastogenesis-inhibitory activity ofthe anti-OBM/sOBM antibodies can also be evaluated by measuring theirbone resorption-inhibitory activity utilizing experimental animals invivo. Especially, ovariectomized animal model is given as an animalmodel with progressive osteoclast formation. Theosteoclastogenesis-inhibitory activity of the anti-OBM/sOBM antibodiescan be determined by administering the anti-OBM/sOBM antibodies to suchexperimental animals and evaluating the suppression of bone resorption(a bone density increasing activity).

The thus-obtained antibodies capable of neutralizing the OBM/sOBMbiological activity are useful in pharmaceutical compositions,particularly pharmaceutical compositions to prevent or treat bonemetabolism abnormality or as antibodies for an immunological diagnosisof such diseases. The preparations comprising the antibodies of thepresent invention can be administered either orally or non-orally. Suchpreparations can be safely administered to humans or animals aspharmaceutical compositions which contain the antibodies recognizing OBMand/or sOBM as an active component. As the forms of pharmaceuticalcomposition, injection agents including intravenous drip, suppositoryagents, sublingual agents, percutaneous absorption agents, and the likeare given. Because monoclonal antibodies are macromolecule proteins,they not only readily adhere to a glass container such as a vial or asyringe, but also are easily denatured by physicochemical factors suchas heat, pH, or humidity. Therefore, the preparations should bestabilized by the addition of stabilizers, pH adjusters, bufferingagents, solubilizing agents, or detergents. As the stabilizers, aminoacids such as glycine and alanine, saccharides such as dextran 40 andmannose, and sugar alcohols such as sorbitol, mannitol, and xylitol canbe given. These stabilizers may be used either individually or incombinations of two or more. The amount of stabilizers to be added ispreferably from 0.01 to 100 times, particularly preferably from 0.1 to10 times, the amount of the antibody. The addition of these stabilizersincreases storage stability of liquid preparations or lyophilizedproducts thereof. Phosphate buffers and citrate buffers are given asexamples of the buffering agents. The buffering agents not only adjustthe pH of the liquid preparations or aqueous solutions obtained byre-dissolving the lyophilized products thereof, but also increasestability and solubility of the antibody. It is desirable to add thebuffering agent in an amount to make from 1 mM to 10 mM concentration ofthe liquid preparation or of the aqueous solution prepared from thelyophilized product. Polysorbate 20, Pluronic F-68, and polyethyleneglycol are given as examples of the detergent. A particularly preferredexample is Polysorbate 80. These detergents may be used eitherindividually or in combinations of two or more. Macromolecule proteinssuch as an antibody easily adhere to glass containers. Adherence tocontainers of the antibody in a liquid preparation or in an aqueoussolution prepared by re-dissolving a lyophilized product can beprevented by adding such detergents at a concentration from 0.001 to1.0%. The preparations comprising the antibodies of the presentinvention can be obtained by adding stabilizers, buffering agents, oragents which prevent adsorption. When the preparations are used asinjection agents for medication or for animals, such injection agentsshould preferably have an osmotic pressure ratio of 1 to 2. The osmoticpressure ratio can be adjusted by increasing or decreasing the amount ofsodium chloride when making the preparations. The amount of an antibodyin a preparation can be suitably adjusted depending on the disease,route of administration, and the like. A dose of a human antibody tohumans may be changed depending on the affinity of the antibody to humanOBM/sOBM, especially, on the dissociation constant (Kd value) to humanOBM/sOBM. The higher the affinity (or the smaller the Kd value), theless the dose to be administered to humans to obtain a certain medicinaleffect. Because a human-type antibody has a long half-life in blood ofabout 20 days, it is sufficient to administer it to humans at a dose ofabout 0.1-100 mg/kg once or more in a 1-30 day period.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of SDS-PAGE of mouse OBM protein of the presentinvention obtained in Example 3.

Explanation of Symbols

(A):

Lane 1: Molecular weight markers

Lane 2: A partially purified sample (Gly-HCl (pH 2.0) elution fraction)obtained from ST2 cells cultured in the presence of active-form vitaminD₃ and dexamethasone.

Lane 3: A partially purified sample (Gly-HCl (pH 2.0) elution fraction)obtained from ST2 cells cultured in the absence of active-form vitaminD₃ and dexamethasone.

(B):

Lane 1: Molecular weight markers

Lane 2: Mouse OBM protein of the present invention after purification byreverse phase high performance liquid chromatography (Example 3)

FIG. 2 shows the result of the binding assay of ¹²⁵I labeled OCIF toosteoblastic stromal cells, ST2, in Example 4.

FIG. 3 shows the osteoclast formation capability of osteoblastic stromalcells ST2 from different generations in Example 5(1).

Explanation of Symbols

1: Ability of ST2 cells from about a 10^(th) subculture to supportosteoclast formation.

2: Ability of ST2 cells from about a 40^(th) subculture to supportosteoclast formation.

FIG. 4 shows change with the passage of time in expression of theprotein of the present invention on the cell membrane of osteoblasticstromal cells cultured in the presence of active-form vitamin D₃ anddexamethasone in Example 5(2).

FIG. 5 shows change with the passage of time in osteoclast formation inthe co-culture system of Example 5(2).

FIG. 6 shows the inhibitory effect on osteoclast formation when treatedwith OCIF for different culture periods during the co-culture period inExample 5(3).

FIG. 7 shows the results of a crosslinking test of ¹²⁵I-labeled OCIFwith the protein of the present invention in Example 6.

Explanation of Symbols

Lane 1: ¹²⁵1-labeled OCIF-CDD1

Lane 2: ¹²⁵1-labeled OCIF-CDD1 crosslinked with ST2 cells

Lane 3: ¹²⁵1-labeled OCIF-CDD1 crosslinked in the presence of 400-foldexcess of unlabeled OCIF

FIG. 8 shows the result of SDS-PAGE in Example 9.

Explanation of Symbols

Lane 1: Proteins of pOBM291-transfected COS-7 cells immunoprecipitatedin the absence of OCIF

Lane 2: Proteins of pOBM291-transfected COS-7 cells immunoprecipitatedin the presence of OCIF

FIG. 9 shows the results of analysis of binding capability of¹²⁵1-labeled OCIF to COS-7 cells transfected with pOBM291 in Example 10.

Explanation of Symbols

Lanes 1 and 2: The amount of the ¹²⁵1-labeled OCIF binding to COS-7cells transfected with pOBM291

Lanes 3 and 4: The amount of the ¹²⁵1-labeled OCIF binding to COS-7cells transfected with pOBM291 in the presence of 400-fold excess ofunlabeled OCIF

FIG. 10 shows the result of a crosslinking test using OCIF labeled with¹²⁵I in Example 11.

Explanation of Symbols

Lane 1: ¹²⁵1-labeled OCIF

Lane 2: ¹²⁵1-labeled OCIF crosslinked with COS-7 cells transfected withpOBM291

Lane 3: ¹²⁵1-labeled OCIF crosslinked with COS-7 cells transfected withpOBM291 in the presence of 400-fold excess of unlabeled OCIF

FIG. 11 shows the result of a Northern Blot in Example 12.

Explanation of Symbols

Lane 1: RNA originating from ST2 cells cultured without addition ofVitamin D and dexamethasone

Lane 2: RNA originating from ST2 cells cultured with the addition ofVitamin D and dexamethasone

FIG. 12 shows the OCIF-binding capability of the proteins in theconditioned medium at various OCIF concentrations in Example 14(2).

Explanation of Symbols

: pCEP4

: pCEP sOBM

FIG. 13 shows the OCIF-binding capability of the protein in theconditioned medium at various proportions of the conditioned medium inExample 14(2).

Explanation of Symbols

: pCEP4

: pCEP sOBM

FIG. 14 shows the result of SDS-PAGE of a fusion protein consisting ofthioredoxin and mouse OBM expressed in Escherichia coli in Example15(2).

Explanation of Symbols

Lane 1: Molecular weight markers

Lane 2: Soluble protein fractions originating from GI724/pTrxFus

Lane 3: Soluble protein fractions originating from GI724/pTrxOBM25

FIG. 15 shows the OCIF-binding capability at various proportions ofsoluble protein fractions in Example 15(3).

Explanation of Symbols

: GI724/pTrxFus

: GI724/pTrxOBM25

FIG. 16 shows the OCIF-binding capability of soluble protein fractions(1%) at various concentrations of OCIF in Example 15(3).

Explanation of Symbols

: GI724/pTrxFus

: GI724/pTrxOBM25

FIG. 17 shows the results of inhibition of specific binding to OCIF ofmouse OBM obtained by expression of mouse OBM cDNA of the presentinvention and purification or natural OCIF-binding protein by a rabbitanti-mouse OBM antibody.

Explanation of Symbols

1: Purified OBM prepared by expression of the cDNA in the presence ofthe antibody, OBM+¹²⁵I—OCIF

2: The natural protein in the presence of the antibody+¹²⁵I—OCIF

3: Mouse OBM prepared by expression of the cDNA in the absence of theantibody, mouse OBM+¹²⁵I—OCIF

4: The natural protein in the absence of the antibody+¹²⁵I—OCIF

5: 3+unlabeled OCIF (400-fold more than ¹²⁵I—OCIF)

6: 4+unlabeled OCIF (400-fold more than ¹²⁵I—OCIF)

FIG. 18 shows the result of SDS-PAGE of human OBM protein expressed bythe cDNA of the present invention.

Explanation of Symbols

Lane 1: Molecular weight markers

Lane 2: Proteins of COS-7 cells transfected with phOBM (an expressionvector containing a cDNA of the present invention), immunoprecipitatedwith a rabbit anti-OCIF polyclonal antibody in the absence of OCIF

Lane 3: Proteins of COS-7 cells transfected with phOBM (an expressionvector containing a cDNA of the present invention), immunoprecipitatedwith a rabbit anti-OCIF polyclonal antibody in the presence of OCIF

FIG. 19 shows the result of analysis of binding of OCIF to COS-7 cellstransfected with phOBM, an expression vector containing a cDNA of thepresent invention.

Explanation of Symbols

Lane 1: COS-7 cells transfected with phOBM and the addition of125I—OCIF.

Lane 2: COS-7 cells transfected with phOBM and the addition of¹²⁵I—OCIF, in the presence of a 400-fold more unlabeled OCIF

FIG. 20 shows the result of crosslinking of human OBM, which is aprotein encoded by a cDNA of the present invention, with ¹²⁵I—OCIF(monomer-type).

Explanation of Symbols

Lane 1: ¹²⁵I—OCIF

Lane 2: The crosslinked products of ¹²⁵I—OCIF with the proteins on themembrane of COS-7 cells transfected with phOBM.

Lane 3: The crosslinked products of ¹²⁵I—OCIF with the proteins on themembrane of COS-7 cells transfected with pHOBM, in the presence of a400-fold more unlabeled OCIF.

FIG. 21 shows the OCIF-binding capability of the protein (secreted-formhOBM) in the conditioned medium at various OCIF concentrations inExample 24(2).

Explanation of Symbols

: Conditioned medium of 293-EBNA cells transfected with pCEP4, whichdoes not contain cDNA encoding secreted-form human OBM

: Conditioned medium of 293-EBNA cells transfected with pCEPshOBM, whichcontains cDNA encoding secreted-form human OBM

FIG. 22 shows the OCIF-binding capability of the protein (secreted-formhuman OBM) in the conditioned medium at a specific OCIF concentrationwhile changing the amount of conditioned medium added in Example 24(2).

Explanation of Symbols

: Conditioned medium of 293-EBNA cells transfected with pCEP4, whichdoes not contain cDNA encoding secreted-form human OBM

: Conditioned medium of 293-EBNA cells transfected with pCEPshOBM, whichcontains cDNA encoding secreted-form human OBM

FIG. 23 shows the result of SDS-PAGE of a fusion protein consisting ofthioredoxin and human OBM expressed in Escherichia coli.

Explanation of Symbols

Lane 1: Molecular weight markers

Lane 2: Soluble protein fractions originating from Escherichia coliGI724/pTrxFus

Lane 3: Soluble protein fractions originating from Escherichia coliGI724/pTrxhOBM

FIG. 24 shows the OCIF-binding capability of the fusion proteinconsisting of thioredoxin and human OBM to OCIF, when the amount of thesoluble protein fraction originating from Escherichia coli including thefusion protein added was varied in Example 25(3).

Explanation of Symbols

: Soluble protein fractions originating from Escherichia coliGI724/pTrxFus

: Soluble protein fractions originating from Escherichia coliGI724/pTrxshOBM

FIG. 25 shows the OCIF-binding capability of the fusion protein ofthioredoxin and human OBM in soluble protein fractions originating fromEscherichia coli to OCIF in various concentrations in Example 25(3).

Explanation of Symbols

: Soluble protein fractions originating from Escherichia coliGI724/pTrxFus

: Soluble protein fractions originating from Escherichia coliGI724/pTrxshOBM

FIG. 26 shows the result of quantifying human OBM and human sOBM by thesandwich ELISA method using the rabbit anti-human OBM/sOBM polyclonalantibody of the present invention.

Explanation of Symbols

: Human OBM

: Human sOBM

FIG. 27 shows the result of quantifying human OBM and human sOBM by thesandwich ELISA method using the anti-human OBM/sOBM monoclonalantibodies of the present invention.

Explanation of Symbols

: Human OBM

: Human sOBM

FIG. 28 shows the result of quantifying mouse OBM and sOBM by thesandwich ELISA method using the anti-human OBM/sOBM monoclonalantibodies of the present invention which cross react mouse OBM andsOBM.

Explanation of Symbols

: Mouse OBM

: Mouse sOBM

FIG. 29 shows the activity of the fusion protein consisting ofthioredoxin and mouse OBM to stimulate human osteoclast-like cellformation.

FIG. 30 shows the suppression of the anti-OBM/sOBM antibody of the boneresorption activity stimulated by vitamin D₃.

FIG. 31 shows the suppression of the anti-OBM/sOBM antibody of the boneresorption activity stimulated by prostaglandin E₂ (PGE₂).

FIG. 32 shows the suppression by the anti-OBM/sOBM antibody of thebone-resorbing activity stimulated by parathyroid hormone (PTH).

FIG. 33 shows the suppression by the anti-OBM/sOBM antibody of thebone-resorbing activity stimulated by interleukin 1 α (1L-1).

BEST MODE FOR CARRYING OUT THE INVENTION Examples

The present invention will be described in more detail by way ofexamples which are given for the purpose of illustration of theinvention and are not limiting thereof in any way of the remainder ofthe disclosure.

Example 1 Preparation of the Protein of the Present Invention (1)Large-Scale Cultivation of ST2 Cells

Mouse osteoblastic stromal cell line ST2 (RIKEN CELL BANK RCB0224) wascultured using α-MEM containing 10% fetal bovine serum. ST2 cellscultured to confluence in a 225 cm² T flask for adherent-cell culturewere treated with trypsin and harvested from the T flask. After washing,the cells were transferred to five 225 cm² T flasks. After the additionof 60 ml of α-MEM containing 10⁻⁸ M active-form vitamin D₃ (Calcitriol),10⁻⁷ dexamethasone, and 10% fetal bovine serum, cells in each flask werecultured for 7-10 days in a CO₂ incubator. The cultured ST2 cells wereharvested using a cell scraper and stored at −80° C. until use.

(2) Preparation of Membrane Fraction and Solubilization ofMembrane-Bound Proteins

To the ST2 cells (volume, about 12 ml) described in Example 1(1),cultured using eighty 225 cm² T flasks, was added three times the volume(36 ml) of 10 mM Tris-HCl buffer (pH 7.2) containing protease inhibitors(2 mM APMSFP, 2 mM EDTA, 2 mM o-phenanthroline, 1 mM leupeptin, 1 μg/mlpepstatin A, and 100 unit/ml aprotinin). After vigorously agitating for30 seconds using a vortex mixer, the cells were allowed to stand for 10minutes on ice. The cells were homogenized using a homogenizer (DOUNCETISSUE GRINDER, A syringe, WHEATON SCIENTIFIC Co.). The same volume (48ml) of 10 mM Tris-HCl buffer (pH 7.2) containing the above-mentionedprotease inhibitors, 0.5 M sucrose, 0.1 M potassium chloride, 10 mMmagnesium chloride, and 2 mM calcium chloride was added to thehomogenized cells. After stirring, the mixture was centrifuged at 600×gfor 10 minutes at 4° C., thereby separating nuclei and non-homogenizedcells as precipitate. The supernatant obtained by the centrifuge wascentrifuged at 150,000×g for 90 minutes at 4° C., to obtain the membranefraction of the ST2 cells as a precipitate. Eight ml of 10 mM Tris-HClbuffer (pH 7.2) containing the above-mentioned protease inhibitors, 150mM sodium chloride, and 0.1 M sucrose was added to this membranefraction. After the addition of 200 μl of 20% CHAPS(3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate, Sigma Co.),the mixture was stirred for 2 hours at 4° C. The mixture was thencentrifuged at 150,000×g for 60 minutes at 4° C., to obtain supernatantas solubilized membrane fraction.

Example 2 Purification of the Protein of the Present Invention (1)Preparation of OCIF-Immobilized Affinity Column

After replacing iso-propanol in a HiTrap NHS-activated column (1 ml,manufactured by Pharmacia Co.) with 1 mM hydrochloric acid, 1 ml of 0.2M NaHCO₃/0.5 M NaCl solution (pH 8.3) containing 13.0 mg of recombinantOCIF prepared by the method of WO 96/26217 was added to the column usinga syringe (5 ml, manufactured by Terumo Corp.), to effect couplingreaction at room temperature for 30 minutes. The column was fed with 3ml of 0.5 M ethanolamine/0.5 M NaCl (pH 8.3) and 3 ml of 0.1 M aceticacid/0.5 M NaCl (pH 4.0) three times in turn to inactivate excess activegroups, then the solution was replaced with 0.5 M ethanolamine/0.5 MNaCl (pH 8.3). After allowing to stand at room temperature for 1 hour,the column was washed twice alternately with 0.5 M ethanolamine/0.5 MNaCl (pH 8.3) and 0.1 M acetic acid/0.5M NaCl (pH 4.0), and the solutionwas then replaced with 50 mM Tris/1 M NaCl/0.1% CHAPS buffer (pH 7.5).

(2) Purification of the Protein of the Present Invention UsingOCIF-Immobilized Affinity Column

The purification of the OCIF-binding protein was carried out at 4° C.,unless otherwise indicated. The above-mentioned OCIF-immobilizedaffinity column was equilibrated with 10 mM Tris-hydrochloride buffer(pH 7.2) to which the protease inhibitors described in Example 1(2),0.15 M sodium chloride, and 0.5% CHAPS were added. About 8 ml of thesolubilized membrane fraction described in Example 1(2) was applied tothe column at a flow rate of 0.01 ml/minute. Then, the column was washedwith 10 mM Tris-hydrochloride buffer (pH 7.2) to which theabove-mentioned protease inhibitors, 0.15 M sodium chloride, and 0.5%CHAPS was added, for 100 minutes at a flow rate of 0.5 ml/minute. Next,the proteins adsorbed to the column was eluted with 0.1 Mglycine-hydrochloride buffer (pH 3.3) containing the proteaseinhibitors, 0.2 M sodium chloride, and 0.5% CHAPS for 50 minutes at aflow rate of 0.1 ml/minute. In the same manner, the proteins adsorbed tothe column was eluted with 0.1 M sodium citrate buffer (pH 2.0)containing the protease inhibitors, 0.2 M sodium chloride, and 0.5%CHAPS for 50 minutes at a flow rate of 0.1 ml/minute. The eluate wascollected in 0.5 ml fractions. Each fraction was immediately neutralizedby the addition of 2M Tris solution. The fractions derived from theelution with these buffers (each fraction consisting of 1.0-5.0 ml ofeluate) were concentrated to 50-100 μl using Centricon-10 (manufacturedby Amicon of U.S.A.). OCIF was added to a portion of each concentratedfraction and immunoprecipitated with anti-OCIF polyclonal antibody. Theprecipitated fractions were treated with SDS and subjected to SDS-PAGE.Fractions (Fr. No. 3-10) in which the band of the protein with specificbinding ability to OCIF appeared were regarded as the protein fractionsof the present invention.

(3) Purification of the Protein of the Present Invention by GelFiltration

The concentrated OCIF-binding protein (the fractions obtain by theelution with 0.1 M glycine-hydrochloride buffer (pH 3.3) and 0.1 Msodium citrate buffer (pH 2.0)) prepared in Example 2(2) was applied toa Superose 12 HR10/30 column (1.0×30 cm, manufactured by Pharmacia Co.)which was equilibrated with 10 mM Tris-HCl, 0.5 M NaCl, 0.5% CHAPS (pH7.0) and developed with the equilibration buffer as a mobile phase at aflow rate of 0.5 ml/min, and each 0.5 ml fraction was collected. Thefractions containing the protein of the present invention (Fr. Nos.27-32) were identified according to the same method as described above.Each of the fractions was concentrated using Centricon-10 (a product ofAmicon).

(4) Purification by Reverse Phase High Performance Liquid Chromatography

The above-mentioned OCIF-binding protein purified by the gel filtrationwas applied to a C₄ column (2.1×250 mm, Vydac, USA) which wasequilibrated with 0.1% trifluoroacetic acid (TFA) and 30% acetonitrile.The proteins bound to the column were eluted with linear gradients ofacetonitrile from 30% to 55% for the first 50 minutes and from 55% to80% during the next 10 minutes at a flow rate of 0.2 ml/min. Peaks ofeluted proteins were detected by measuring optical density at 215 nm.Proteins in the different peaks were analyzed to identify the fractionscontaining the protein of the present invention, and a highly purifiedprotein of the present invention was obtained.

Example 3 SDS-PAGE of the Purified Protein of the Present Invention

The solubilized membrane fraction prepared from ST2 cells which werecultured in the presence or absence of active-form vitamin D₃ wassubjected to purification with the OCIF-immobilized affinity column. Thepurified preparations were subjected to SDS-PAGE. As shown in FIG. 1(A),a major protein band with MW of about 30,000-40,000 was detected only inthe purified preparation from ST2 cells which was cultured in thepresence of the active-form vitamin D₃, proving that the protein whichspecifically binds to OCIF (i.e. the protein of the present invention)can be selectively purified by the OCIF-immobilized affinity column.However, bands of several proteins (other than the protein of thepresent invention) which non-specifically bind to carriers or spacers ofthe OCIF-immobilized column were detected in both of the purifiedpreparations. The proteins other than the protein of the presentinvention were removed according to the above-described method by gelfiltration and C4 reverse phase chromatography. SDS-PAGE of the obtainedhighly purified protein of the present invention is shown in FIG. 1(B).The highly purified protein of the present invention was found to beelectrophoretically homogeneous and had a molecular weight of about30,000-40,000.

Example 4 Binding Test of OCIF to Osteoblasts (1) Preparation of¹²⁵I-Labeled OCIF

OCIF was labeled with ¹²⁵I by the Iodogen method. Specifically, 20 μl of2.5 mg/ml Iodogen-chloroform solution was transferred to a 1.5 mlEppendorf tube and chloroform was evaporated off at 40° C., to obtain atube coated with Iodogen. The tube was washed three times with 400 μl of0.5 M sodium phosphate buffer (Na-Pi, pH 7.0). Five μl of 0.5 M Na-Pi(pH 7.0) was added to the tube. Immediately after the addition of 1.3 μl(18.5 MBq) of Na-¹²⁵I solution (NEZ-033H20, manufactured by AmershamCo.); 10 μl of 1 mg/ml rOCIF solution (monomer type or dimer type) wasadded to the tube. After mixing with a vortex mixer, the mixture wasallowed to stand at room temperature for 30 seconds. The solution wastransferred to a tube containing 80 μl of a solution of 10 mg/mlpotassium iodide in 0.5 M Na-Pi (pH 7.0) and 5 μl of a phosphatebuffered saline containing 5% bovine serum albumin, and stirred. Themixture was applied to a spin column (1 ml, G-25 fine, manufactured byPharmacia Co.) which was equilibrated with phosphate buffered salinecontaining 0.25% bovine serum albumin and the column was centrifuged for5 minutes at 2,000 rpm. Four hundred μl of a phosphate buffered salinecontaining 0.25% bovine serum albumin was added to the fraction elutedfrom the column and the mixture was stirred. Two of the aliquot wasremoved to measure the radioactivity using a gamma counter. Theradiochemical purity of the ¹²⁵I-labeled OCIF was determined bymeasuring the radioactivity precipitated with 10% TCA. The biologicalactivity of the ¹²⁵I-labeled OCIF was measured according to the methoddescribed in WO 96/26217. The concentration of the ¹²⁵I-labeled OCIF wasmeasured by the ELISA according to the following procedure.

(2) Measurement of the Concentration of ¹²⁵I-Labeled OCIF by ELISA

One hundred μl of 50 mM NaHCO₃ (pH 9.6) in which the anti-OCIF rabbitpolyclonal antibody described in WO 96/26217 was dissolved to aconcentration of 2 μg/ml was added to each well of a 96-wellimmuno-plate (MaxiSorp™, a product of Nunc Co.). The plate was allowedto stand overnight at 4° C. After removing the solution by suction, 300μl of Block Ace™ (Snow Brand Milk Products Co., Ltd.)/phosphate bufferedsaline (25/75) solution was added to each well. The plate was thenallowed to stand for two hours at room temperature. After removing thesolution by suction, the wells were washed three times with phosphatebuffered saline containing 0.01% Polysorbate 80 (P—PBS). Next, 300 μl ofBlock Ace™/phosphate buffered saline (25/75) solution to which¹²⁵I-labeled OCIF or the standard OCIF preparation was mixed, was addedto each well. The plate was then allowed to stand for two hours at roomtemperature. After removing the solution by suction, each well waswashed six times with 200 μl of P—PBS.

One hundred μl of Block Ace™ (Snow Brand Milk Products Co.,Ltd.)/phosphate buffered saline (25/75) solution containing peroxidaselabeled rabbit anti-OCIF polyclonal antibody was added to each well. Theplate was allowed to stand for two hours at room temperature. Afterremoving the solution by suction, the wells were washed six times with200 μl P—PBS. Then, 100 μl of TMB solution (TMB Soluble Reagent, HighSensitivity, Scytek Co.) was added to each well. After incubating atroom temperature for 2-3 minutes, 100 μl of stopping solution (StoppingReagent, Scytek Co.) was added to each well. Absorbance of each well wasmeasured at 490 nm using a microplate reader. The concentration of¹²⁵I-labeled OCIF was determined from a calibration curve prepared usingthe standard preparation of OCIF.

(3) Binding Test of OCIF to Osteoblasts or Spleen Cells

Mouse osteoblastic stromal cell line ST2 or spleen cells were suspendedin α-MEM containing 10% fetal bovine serum (FBS), either with or without10⁻⁸ M active-form vitamin D₃ (Calcitriol) and 10⁻⁷ M dexamethasone, toa concentration of 4×10⁴ cells/ml (ST2 cells) or 2×10⁶ cells/ml (spleencells), respectively. Each cell suspension was innoculated into a24-well micro plate. The cells were cultured for 4 days in a CO₂incubator. After washing the cells with α-MEM, 200 μl of medium for thebinding test (α-MEM to which 0.2% bovine serum albumin, 20 mM Hepesbuffer, and 0.2% NaN₃ were supplemented), containing 20 ng/ml ofabove-described ¹²⁵I-labeled OCIF (monomer form or dimer form), wasadded to each well. To the wells for the measurement of non-specificbinding, 200 μl of the medium for the binding test containing 8 μg/ml ofrOCIF (400 times concentration) in addition to 20 ng/ml of ¹²⁵I-labeledOCIF was added. The cells were cultured for one hour in a CO₂ incubatorand washed 3 times with 1 ml of a phosphate buffered saline. In thisprocedure, spleen cells were washed by centrifuging the 24-well plate ineach washing step, because the spleen cells were non-adherent. Afterwashing, 500 μl of 0.1 N NaOH solution was added to each well and theplate was allowed to stand for 10 minutes at room temperature todissolve the cells. The amount of RI in each well was measured by agamma counter.

As shown in FIG. 2, ¹²⁵I-labeled OCIF did not bind to the culturedspleen cells, but specifically bound only to the osteoblastic stromalcells which were cultured in the presence of active-form vitamin D₃. Theresults indicated that the protein of the present invention is amembrane bound protein induced by active-form vitamin D₃ anddexamethasone on osteoblastic stromal cells.

Example 5 Biological Activity of the Protein of the Present Invention(1) Osteoclasts-Formation Supported by Osteoblastic Stromal Cells

The osteoclasts formation-supporting capability of osteoblastic stromalcells was evaluated by measuring tartaric acid resistant acidphosphatase activity (TRAP activity) of the formed osteoclasts.Specifically, spleen cells (2×10⁵ cells/100 μl/well) from a ddy mouse(8-12 weeks old) and mouse osteoblastic stromal cells ST2 (5×10³cells/100 μl/well) were suspended in α-MEM to which 10⁻⁸ M active-formvitamin D₃, 10 M dexamethasone, and 10% fetal bovine serum were added.The cells were innoculated into 96-well plates and cultured for one weekin a CO₂ incubator. After washing each well with phosphate bufferedsaline, 100 μl of ethanol/acetone (1:1) was added to each well, and thecells were immobilized at room temperature for one minute. Afterimmobilization, 100 μl of 50 mM citrate buffer (pH 4.5) containing 5.5mM p-nitrophenol phosphate and 10 mM sodium tartarate was added to eachwell. After 15 minutes of reaction at room temperature, 0.1 N NaOHsolution was added to each well and absorbance at 405 nm was measuredusing a microplate reader. The results of osteoclasts-formation by ST2cells with a passage number of about 10 or 40 after purchasing the cellsfrom RIKEN CELL BANK are shown in FIG. 3. The results indicate that theST2 cells with a higher passage number exhibit more potent ability tosupport osteoclasts-formation.

(2) Time Course Change of Expression of the Protein of the PresentInvention on Membrane of Osteoblastic Stromal Cells in a Culture Systemwhich Include Active-Form Vitamin D₃ and Dexamethasone and Time CourseChange of Osteoclasts-Formation in the Co-Culture System

In the same manner as in Example 4(3), osteoblastic stromal cell ST2 wascultured for 7 days in the presence of active-form vitamin D₃ anddexamethasone. The OCIF-binding test was carried out using ¹²⁵I-labeledOCIF (monomer type) as described in the experiment in Example 4(1).Non-specific binding was measured by competing ¹²⁵I—OCIF binding to ST2cells with 400-fold concentration of unlabeled OCIF. As a result, it wasconfirmed that the amount of specific binding of ¹²⁵I-labeled OCIFincrease in accordance with increase in culture period in the presenceof active-form vitamin D₃ and dexamethasone. Specifically, as shown inFIGS. 4 and 5, the protein of the present invention was expressed on thesurface of ST2 cells by active-form vitamin D₃ in accordance withincrease in culture period and the expression reached a maximum on thefourth day of culture. On the other hand, osteoclast-like cells areformed by coculturing mouse spleen cells and ST2 cells in the presenceof active-form vitamin D₃. TRAP (a marker enzyme ofosteoclasts)-positive mononuclear pre-osteoclast-like cells are formedon the third or fourth day of the culture. More differentiated andmature TRAP-positive multinuclear cells are formed on the fifth to sixthday of the culture. A good correlation between time-course of theexpression of the protein of the present invention andosteoclasts-formation was thus demonstrated.

(3) Inhibition of Osteoclasts Formation by OCIF Treatment for DifferentPeriod During the Co-Culture

To make it clear that the protein of the present invention is a factorinvolved in the osteoclasts-formation, the cells were treated with 100mg/ml OCIF for different culture periods during the six day co-cultureperiod described in the above-mentioned Example 5(2) (two consecutivedays in the six-day period, except for the 5th day for which a one-dayperiod was applied). As a result, as shown in FIG. 6, OCIF treatment at48-96 hours after start of the culture at which expression of theprotein of the present invention on ST2 cells is maximal was found to bemost effective for inhibiting formation of osteoclasts. Specifically, itwas confirmed that OCIF controls osteoclast formation by binding to ST2cells via the protein of the present invention.

Based on the results of the above experiments, the protein of thepresent invention was confirmed to be induced on cell membrane ofosteoblastic stromal cells by active-form vitamin D₃ and dexamethasoneand to exhibit a biological activity to support or acceleratedifferentiation or maturation of osteoclasts.

Example 6 Crosslinking Test for ¹²⁵I-Labeled OCIF and the Protein of thePresent Invention

To identify the protein of the present invention more clearly, theprotein of the present invention was crosslinked with ¹²⁵I-labeled OCIF.Mouse osteoblastic stromal cell line ST2 was cultured for four days inthe presence or absence of active-form vitamin D₃ and dexamethasone inthe same manner as described in Example 4(3). After washing the cellswith 1 ml of phosphate buffered saline, 200 μl of medium for bindingtest (α-MEM to which 0.2% bovine serum albumin, 20 mM Hepes buffer, 0.2%NaN₃, and 100 μg/ml heparin were added), containing 25 ng/ml of¹²⁵I-labeled OCIF (monomer type) or 40 ng/ml of ¹²⁵I-labeled OCIF-CDD1which was obtained by expressing the protein of Sequence ID No. 76 (WO96/26217) in animal cells, was added. The above-mentioned culture mediumfor the binding test was further supplemented with 400-foldconcentration of OCIF and was added to the other wells to assessnon-specific binding. After culturing for one hour in a CO₂ incubator,each well was washed three times with 1 ml of phosphate buffered salinecontaining 100 μg/ml heparin. Five hundred μl of phosphate bufferedsaline containing 100 μg/ml crosslinking agent, DSS (Disuccinimidylsuberate, Pierce Co.), was added to each well and the plate was kept for10 minutes at 0° C. The wells were washed twice with 1 ml of phosphatebuffered saline at 0° C. One hundred 111 of 20 mM Hepes buffercontaining 1% Triton X-100, 10 μM pepstatin, 10 μM leupeptin, 2 mM PMSF(phenylmethylsulfonyl fluoride), 10 μM antipain, and 2 mM EDTA, was thenadded to each well. The plate was allowed to stand for 30 minutes atroom temperature to dissolve the cells. Fifteen μl of these samples weretreated with SDS under non-reducing conditions according to conventionalmethod and subjected to SDS-polyacrylamide gel electrophoresis (4-20%polyacrylamide gradient, manufactured by Daiichi Chemical Co., Ltd.).After electrophoresis, the gels were dried and exposed to BioMax MS film(manufactured by Kodak) for 24 hours at −80° C. using BioMax MSintensifying screens (manufactured by Kodak). After exposure, the filmwas developed by conventional method. A band of crosslinking productwith a molecular weight of 90,000-110,000 was detected when the¹²⁵I-labeled OCIF (monomer type, 60 kDa) was used. When the ¹²⁵I-labeledOCIF-CDD1 (31 kDa) was used, a band of crosslinking product of about70-80 kDa (average, 78 kDa) was detected as shown in FIG. 7.

Example 7 Analysis of the Protein of the Present Invention Expressed onST Cells by Scatchard Plot

The above-mentioned ¹²⁵I-labeled OCIF (monomer type) was added to aconcentration of 1,000 μM to the culture medium for binding test (α-MEMcontaining 0.2% bovine serum albumin, 20 mM Hepes buffer, and 0.2% NaN₃)and the culture medium was serially diluted at a rate to ½ with theculture medium not containing ¹²⁵I-labeled OCIF. Solutions for measuringnon-specific binding were prepared by further adding 400-foldconcentration of monomer-form OCIF to these solutions. Two hundred μl ofthe prepared solutions were added to the above-mentioned-wells with ST2cells cultured for 4 days (passage number, about 10) in the presence of10⁻⁸ M active-form vitamin D₃ (Calcitriol) and 10⁻⁷ M dexamethasone, toassess binding of ¹²⁵I-labeled OCIF in the same method as described inExample 4(3). The results were subjected to Scatchard Plot analysis todetermine the dissociation constant of OCIF and OCIF-binding protein andthe number (site) of OCIF-binding protein per a ST2 cell. As a result,the dissociation constant of OCIF and the protein of the presentinvention was found to be 280 μM, and the number of the site ofOCIF-binding protein per a ST2 cell was approximately 33,000/cell. Basedon the finding in Example 5(1) that osteoclasts-formation supported bythe ST2 cells with passage number about 40 was more extensive than thatwith passage number about 10, the number (the site) of the protein ofthe present invention expressed on the ST2 cell with a passage numberabout 40 was assessed. The number (site) was 58,000/cell and was clearlylarger than the ST2 cells with passage number about 10, indicating thatthe amount of the protein of the present invention expressed on ST2cells is related to their potency to support osteoclasts-formation. Theresults indicated that the protein of the present invention is a factorthat supports or induces differentiation or maturation of osteoclasts.

Example 8 Cloning of OBM cDNA

(1) Extraction of RNA from Mouse ST2 Cells

Mouse osteoblastic stromal cell line ST2 (RIKEN CELL BANK, RCB0224) wascultured in α-MEM (Gibco BRL Co.) containing 10% fetal bovine serum. ST2cells cultured to confluent in a 225 cm² T-flask for adherent cells weretreated with trypsin to harvest the cells from the T-flask. The cellswere washed and transferred to five 225 cm² T-flasks. Sixty ml of α-MEMcontaining 10⁻⁸ M active-form vitamin D₃ (Calcitriol, Wako PureChemicals Co., Ltd.), 10⁻⁷ M dexamethasone, and 10% fetal bovine serumwas added to each flask and the cells were cultured for 5 days in a CO₂incubator. Total RNA was extracted from the cultured ST2 cells usingISOGEN (Wako Pure Chemicals Co., Ltd.). PolyA⁺ RNA was prepared fromabout 600 μg of the total RNA using an Oligo(dT)-cellulose column (5′-3′Prime Co.). About 8 μg of Poly A⁺ RNA was obtained.

(2) Construction of Expression Library

Double-stranded cDNA was synthesized from 2 μg of polyA⁺ RNA obtained inExample 8(1) using a Great Lengths cDNA Synthesis kit (Clontech Co.)according to the instruction in the manual. Specifically, 2 μg of polyA⁺RNA and Oligo(dT)₂₅(dN) primer were mixed and distilled water was addedto the mixture to make the final volume to 6.25 μl. After incubation forabout 3 minutes at 70° C., the mixture was cooled on ice for 2 minutes.To the mixture were added 2.2 μl of distilled water, 2.5 μl of 5×First-strand buffer, 0.25 μl of 100 mM DTT (dithiothreitol), 0.5 μl ofPRIME RNase inhibitor (1 U/ml) (5′-3′ Prime Co.), 0.5 μl of [α-³²P]dCTP(Amersham Co., 3000 Ci/mmol) diluted 5-fold with distilled water to make2 μCi/μl, 0.65 μl of dNTP (20 mM each), and 1.25 μl (250 unit) ofMMLV(RNaseH⁻) reverse transcriptase. The mixture was incubated for 90minutes at 42° C., followed by the further addition of 62.25 μl ofdistilled water, 20 μl of 5× second-strand buffer, 0.75 μl of dNTP (20mM each), and 5 μl of Second-strand enzyme cocktail. The resultingmixture was maintained at 16° C. for two hours. Then, 7.5 units of T4DNA polymerase was added to this reaction mixture. After incubation at16° C. for 30 minutes, the reaction was terminated by the addition of 5μl of 0.2M EDTA. After a phenol-chloroform treatment, the product wasprecipitated with ethanol. An EcoRI-SalI-NotI linker (Clontech Co.) wasattached to the ends of the resultant double-stranded cDNA. Then, theends were phosphorylated and the product was applied on a sizefractionation column to obtain cDNA with a length more than 500 bp. DNAwas precipitated with ethanol, dissolved in water and ligated to pcDL-SRα296 (Molecular and Cellular Biology, Vol. 8, pp 466-472, 1988) whichhad been cut with a restriction enzyme EcoRI (Takara Shuzo Co.) andtreated with CIAP (calf intestine alkaline phosphatase, Takara ShuzoCo.).

(3) Screening of Expression Library by Means of Binding to OCIF

An escherichia coli strail, XL2 Blue MRF′ (Toyobo Co., Ltd.), wastransformed using the DNA produced in Example 8(2) and cultured onL-Carbenicillin agar (1% tryptone, 0.5% yeast extract, 1% NaCl, 60 μg/mlcarbenicillin, 1.5% agar) prepared in a 24-well plastic plates, toproduce about 100 colonies per well. Transformants in each well weresuspended in 3 ml of Terrific Broth ampicillin culture medium (1.2%tryptone, 2.4% yeast extract, 0.4% glycerol, 0.017 M KH₂PO₄, 0.072 MK₂HPO₄, 100 μg/ml ampicillin) and cultured at 37° C. overnight withshaking. Cells were collected by centrifugation to prepare plasmid DNAusing a QIAwell kit (QIAGEN Co.). DNA concentration was determined bymeasuring absorbance at 260 nm. DNA was concentrated by precipitatingwith ethanol and dissolved in distilled water to a concentration of 200ng/μl. Five hundred DNA pools, each of which was obtained from about 100colonies were prepared and were used for transfection into COS-7 cells(RIKEN CELL BANK, RCB0539). COS-7 cells were seeded into DMEM containing10% fetal bovine serum in each well of 24-well plates at a cell densityof 8×10⁴ cells/well and cultured overnight at 37° C. in a CO₂ incubator.Next day, the culture medium was removed and the cells were washed withserum-free DMEM culture medium. The above-described plasmid DNA whichwas previously diluted with an OPTI-MEM culture medium (Gibco BRL Co.)and mixed with Lipofectamine (a transfection reagent, manufactured byGibco BRL Co.) according to the protocol supplied with Lipofectamine.After 15 minutes, the mixture was added to the cells in each well. Theamount of Lipofectamine and DNA used were, respectively, 1 μg and 4 μlper well. After 5 hours, the culture medium was removed and 1 ml of DMEMculture medium (Gibco BRL Co.) containing 10% fetal bovine serum wasadded to each well. The plates were incubated for 2-3 days at 37° C. ina CO₂ incubator (5% CO₂). The COS-7 cells transfected and cultured for2-3 days in this manner were washed with a serum-free DMEM culturemedium. Then, 200 μl of a culture medium for the binding assay(serum-free DMEM culture medium containing 0.2% calf serum albumin, 20mM Hepes buffer, 0.1 mg/ml heparin, and 0.02% NaN₃) with 20 ng/ml of¹²⁵I-labeled OCIF added thereto was added to each well. After culturingfor one hour at 37° C. in a CO₂ incubator (5% CO₂), the cells werewashed twice with 500 μl of a phosphate buffered saline containing 0.1mg/ml heparin. After washing, 500 μl of 0.1 N NaOH solution was added toeach well. The plates were allowed to stand for 10 minutes at roomtemperature to lyse the cells. The amount of ¹²⁵I in each well wasmeasured using a gamma counter (Packard Co.). One DNA pool containingcDNA encoding the protein which specifically binds to OCIF was found byscreening a total of 500 pools. The DNA pool containing the cDNA wasfurther divided, and the above-described transfection and screeningoperations were repeated to isolate the cDNA which encodes the proteinwhich binds to OCIF. The plasmid containing this cDNA was named pOBM291.The Escherichia coli containing this plasmid was deposited with TheNational Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology, Biotechnology Laboratory, as pOBM291on May 23, 1997 under the deposition No. FERM BP-5953.

The methods of labeling OCIF with ¹²⁵I and quantitative analysis of the¹²⁵I-labeled OCIF by ELISA are shown below. Labeling of OCIF with ¹²⁵Iwas carried out according to the Iodogen method. Twenty μl of 25 mg/mlIodogen-chloroform solution was added to a 1.5 ml Eppendorf tube andchloroform was evaporated by heating at 40° C., to prepare anIodogen-coated tube. The tube was washed three times with 400 μl of 0.5M sodium phosphate buffer (Na-Pi, pH 7.0), and 5 μl of 0.5 M Na-Pi (pH7.0) was added. Immediately after the addition of 1.3 μl (18.5 MBq) ofNa—¹²⁵I solution (NEZ-033H20, Amersham Co.), 10 μl of 1 mg/ml rOCIFsolution (monomer type or dimer type) was added to the tube. Aftermixing the contents with a vortex mixer, the tube was allowed to standat room temperature for 30 seconds. The solution in the tube wastransferred to a tube to which 80 μl of 10 mg/ml potassium iodide, 0.5 MNa-Pi (pH 7.0) and 5 μl of a phosphate buffered saline containing 5%bovine serum albumin (BSA-PBS) were previously added. After stiffing,the mixture was applied to a spin column (1 ml, G-25 fine, manufacturedby Pharmacia Co.) equilibrated with BSA-PBS, and the column wascentrifuging for 5 minutes at 2000 rpm. Four hundred μl of BSA-PBS wasadded to the fraction eluted from the column. After stirring, 2 μl of analiquot of this solution was sampled to measure the radioactivity by agamma counter. The radiochemical purity of the ¹²⁵I-labeled OCIFsolution thus prepared was determined by measuring radioactivityprecipitated by 10% TCA. The biological activity of the ¹²⁵I-labeledOCIF was measured according to the method of WO 96/26217. Theconcentration of the ¹²⁵I-labeled OCIF was determined by the ELISA asfollows. Specifically, 100 μl of 50 mM NaHCO₃ (pH 9.6) in which theanti-OCIF rabbit polyclonal antibody described in WO 96/26217 wasdissolved to a concentration of 2 μg/ml was added to each well of a96-well immuno-plate (MaxiSorpa™, a product of Nunc Co.). The plate wasallowed to stand over night at 4° C. After removing the solution bysuction, 300 μl of Block Ace™ (Snow Brand Milk Products Co.,Ltd.)/phosphate buffered saline (25/75) (B—PBS) was added to each well.The plate was then allowed to stand for two hours at room temperature.After removing the solution by suction, the wells were washed threetimes with phosphate buffered saline containing 0.01% Polysorbate80(P—PBS). Next, 100 μl of B—PBS containing ¹²⁵I-labeled OCIF orstandard OCIF was added to each well. The plate was then allowed tostand for two hours at room temperature. After removing the solution bysuction, each well was washed six times with 200 μl of P—PBS. Onehundred μl of peroxidase-labeled rabbit anti-OCIF polyclonal antibodydiluted with B—PBS was added to each well. The plate was allowed tostand for two hours at room temperature. After removing the solution bysuction, the wells were washed six times with 200 μl of P—PBS. Then, 100μl of TMB solution (TMB Soluble Reagent, High Sensitivity, Scytek Co.)was added to each well. After incubating the plate at room temperaturefor 2-3 minutes, 100 μl of stopping solution (Stopping Reagent, ScytekCo.) was added to each well. Absorbance at 450 nm of each well wasmeasured using a microplate reader. The concentration of ¹²⁵I-labeledOCIF was determined based on a calibration curve drawn using thestandard preparation of OCIF.

(4) Determination of the Nucleotide Sequence of the cDNA Encoding theEntire Amino Acid Sequence of OBM

The nucleotide sequence of the OBM cDNA obtained in the Example 8(3) wasdetermined using a Taq DyeDeoxy Terminator Cycle Sequencing kit (aproduct of Perkin Elmer Co.). Specifically, the nucleotide sequence ofthe insert fragment was directly determined using pOBM291 as a template.Fragments with a length of about 1.0 kb and 0.7 kb which were obtainedby digesting pOBM291 with a restriction enzyme EcoRI were inserted intothe EcoRI site of plasmid pUC19 (Takara Shuzo Co.) The nucleotidesequences of these fragments were also determined. The following primerswere used: primer SRR2 which was used to determine nucleotide sequencesof DNA fragments inserted into pcDL-SR α296, M13PrimerM3 and M13PrimerRV(both manufactured by Takara Shuzo Co.) which were used to determine thenucleotide sequences of DNA fragments inserted into plasmid pUC19, andsynthesized primer OBM#8 designed based on the nucleotide sequence ofOBM cDNA. Sequences of these primers are shown as the SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

In addition, the nucleotide sequence of OBM cDNA is shown as SEQ ID NO:2and the amino acid sequence determined therefrom is shown as the SEQ IDNO:1.

Example 9 Expression of the Protein Encoded by the cDNA of the PresentInvention

Plasmid pOBM291 was transfected into COS-7 cells in each well of a6-well plate using Lipofectamine and the transfected COS-7 cells werecultured for two days in DMEM containing 10% fetal bovine serum. Themedium was replaced with a cysteine-methionine-free DMEM (DainipponSeiyaku Co. Ltd.) (800 μl/well) containing 5% dialyzed fetal bovineserum. The cells were cultured for 15 minutes, followed by the additionof 14 of Express Protein Labeling Mix (10 mCi/ml, manufactured by NENCo.). After culturing for four hours, 200 μl of DMEM including 10% fetalbovine serum was added. After one hour culturing, the cells were washedtwice with PBS. Then, 0.5 ml of a TSA buffer (10 mM Tris-HCl (pH 8.0)containing 0.14 M NaCl, 0.025% NaN₃), containing 1% Triton X-100, 1%bovine hemoglobin, 10 μg/ml leupeptin, 0.2 TIU/ml aprotinin, 1 mM PMSF,was added and the mixture was allowed to stand for one hour on ice.Cells were disrupted by pipetting and centrifuged at 3000×g for 10minutes at 4° C. to obtain supernatant. 200 μl of dilution buffer (TSAbuffer containing 0.1% Triton X-100, 0.1% bovine hemoglobin, 10 μg/mlleupeptin, 0.2 TIU/ml aprotinin, 1 mM PMSF) was added to 100 μl of thissupernatant. The mixture was shaken for one hour at 4° C. with protein ASepharose (50 μl) The resultant mixture was centrifuged at 1500×g forone minute at 4° C. to collect supernatant, and thereby fraction(s)which is non-specifically adsorbed to Protein A Sepharose was removed.OCIF (1 μg) was added to this supernatant and the mixture was shaken at4° C. for one hour to achieve the binding of OCIF to OBM. Anti-OCIFpolyclonal antibody (50 μg) was added and the mixture was shaken for onehour at 4° C. Then, Protein A Sepharose (10 μl) was added and themixture was shaken for an additional hour at 40° C., followed bycentrifuge at 1500×g for 1 minute at 40° C. to collect precipitate. Theprecipitate was washed twice with dilution buffer, twice with a bovinehemoglobin-free dilution buffer, once with TSA buffer, and once with 50mM Tris-HCl (pH 6.5). After washing, SDS buffer (0.125 M Tris-HCl, 4%sodium dodecylsulfate, 20% glycerol, 0.002% Bromophenol Blue, pH 6.8)containing 10% β-mercaptoethanol was added to the precipitate. Themixture was heated for 5 minutes at 100° C. and subjected to SDS-PAGE(12.5% polyacrylamide gel, Daiichi Chemical Co., Ltd.). The gel wasfixed according to a conventional method. Isotope signals were amplifiedusing Amplify™ (Amersham Co.) and the sample was exposed to Bio Max MRfilm (KODAK Co.) at −80° C. The results are shown in FIG. 8, whichindicates that the protein encoded by the cDNA of the present inventionhas a molecular weight of about 40,000.

Example 10 Binding of the Protein Encoded by the cDNA of the PresentInvention to OCIF

Plasmid pOBM291 was transfected into COS cells in each well of a 24-wellplate using Lipofectamine. After culturing for 2-3 days, the cells werewashed with serum-free DMEM culture medium. 200 μl of culture medium forthe binding assay (serum-free DMEM culture medium containing 0.2% calfserum, albumin, 20 mM Hepes, 0.1 mg/ml heparin, and 0.2% NaN₃),supplemented with 20 ng/ml ¹²⁵I-labeled OCIF, was added to the wells. Tothe other wells, 200 μl of culture medium for the binding assay to which8 μg/ml of unlabelled OCIF had been added, in addition to 20 ng/ml¹²⁵I-labeled OCIF, was added. After culturing for one hour at 37° C. ina CO₂ incubator (5% CO₂), the cells were washed twice with 500 μl ofphosphate buffered saline containing 0.1 mg/ml of heparin. Then, 500 μlof 0.1 N NaOH solution was added to each well and the plate was allowedto stand for 10 minutes at room temperature to dissolve the cells. Theamount of ¹²⁵I in each well was measured by a gamma counter. As aresult, as shown in FIG. 9, the ¹²⁵I-labeled OCIF was found to bind onlyto the cells in which plasmid pOBM291 was transfected. In addition, thebinding was confirmed to be conspicuously inhibited by the addition of(unlabeled) OCIF at a 400-fold concentration. These results havedemonstrated that the protein OBM encoded by the cDNA on plasmid pOBM291specifically binds to OCIF on the surface of the transfected COS-7cells.

Example 11 Crosslinking of ¹²⁵I-Labeled OCIF and the Protein Encoded bythe cDNA of the Present Invention

Crosslinking of ¹²⁵I-labeled monomer type OCIF and the protein encodedby the cDNA of the present invention was carried out to investigate thecharacteristics of the protein encoded by the cDNA of the presentinvention in further detail. After transfection of plasmid pOBM291 intoCOS-7 cells according to the method used in the Example 8(3), 200 of theculture medium for the binding assay, as described above, containing¹²⁵I-labeled OCIF (25 ng/ml) was added to the wells. The culture mediumfor the binding assay to which unlabeled OCIF at a 400-foldconcentration was added in addition to ¹²⁵I-labeled OCIF was added tothe other wells. After culturing for one hour at 37° C. in a CO₂incubator (5% CO₂), the cells were washed twice with 500 μl of phosphatebuffered saline containing 0.1 mg/ml heparin. Five hundred μl ofphosphate buffered saline containing 100 μg/ml of a crosslinking agent,DSS (disuccinimidyl suberate, manufactured by Pierce Co.) was added tothe cells, followed by a reaction for 10 minutes at 0° C. The cells inthese wells were washed twice with 1 ml of cold phosphate bufferedsaline (0° C.). After the addition of 100 μl of 20 mM Hepes buffercontaining 1% Triton X-100 (Wako Pure Chemicals Co., Ltd.), 2 mM PMSF(Phenylmethylsulfonyl fluoride, Sigma Co.), 10 μM Pepstatin (Wako PureChemicals Co., Ltd.), 10 μM Leupeptin (Wako Pure Chemicals Co., Ltd.),10 μM antipain (Wako Pure Chemicals Co., Ltd.) and 2 mM EDTA (Wako PureChemicals Co., Ltd.), the wells were allowed to stand for 30 minutes atroom temperature to dissolve the cells. Fifteen μl aliquots of thesesamples were heated in the presence of SDS under reducing conditionsaccording to a conventional method and subjected to SDS-electrophoresisusing 4-20% polyacrylamide gradient gel (Daiichi Pure Chemical Co.,Ltd.). After the electrophoresis, the gel was dried and exposed for 24hours at −80° C. to a BioMax MS film (Kodak Co.) using a BioMax MSsensitization screen (Kodak Co.). The exposed film was developedaccording to a conventional method. As a result, a band with a molecularweight of a range of 90,000-110,000, shown in FIG. 10, was detected bycrosslinking the ¹²⁵I-labeled monomer type OCIF and the protein encodedby the cDNA of the present invention.

Example 12 Northern Blotting Analysis

ST2 cells cultured to become confluent in a 25 cm² T flask forattached-cell cultures were treated with trypsin and stripped from the Tflask. After washing, the cells were seeded into a 225 cm² T flask andcultured for 4 days in a CO₂ incubator with 60 ml of an α-MEM culturemedium containing 10⁻⁸ M active-form vitamin D₃, 10 M dexamethasone, and10% fetal bovine serum. Total RNA was extracted from the cultured ST2cells using ISOGEN (Wako Pure Chemicals Co., Ltd.). The total RNA wasalso extracted in the same manner from ST2 cells which were cultured inthe absence of the active-form vitamin D₃ and dexamethasone. After theaddition of 2.0 μl of 5× gel electrophoresis buffer solution (0.2 Mmorpholino propane sulfonic acid, pH 7.0, 50 mM sodium acetate, 5 mMEDTA) and 3.5 μl of formaldehyde, and 10.0 μl of formamide to 20 μg (4.5μl) of each of the total RNAs, the mixtures were incubated for 15minutes at 55° C. and subjected to electrophoresis. The gel forelectrophoresis was prepared according to the formulation of 1.0%agarose, 2.2 M deionized formaldehyde, 40 mM morpholinopropane sulfonicacid (pH 7.0), 10 mM sodium acetate, and 1 mM EDTA. The electrophoresiswas carried out in a buffer solution of 40 mM morpholino propanesulfonic acid, pH 7.0, 10 mM sodium acetate, and 1 mM EDTA. After theelectrophoresis, RNA was transferred onto nylon membrane. About 1.0 kbDNA fragment was obtained by digesting pOBM291 with a restrictionenzyme, EcoRI. Hybridization was carried out using this DNA fragment,labeled with a Megaprime DNA labeling kit (Amersham Co.) and α-³²P-dCTP(Amersham Co.), as a probe. As a result, as shown in FIG. 11, it wasconfirmed that when ST2 cells were cultured in the presence ofactive-form vitamin D₃ and dexamethasone, gene expression of the proteinencoded by the cDNA of the present invention (OBM) is induced strongly.

Example 13 Osteoclasts Formation Supporting Capability of the ProteinEncoded by the cDNA of the Present Invention

pOBM291 was transfected into COS cells according to the same methoddescribed in the Example 8(3). After three days, trypsinized cells werewashed once with phosphate buffered saline solution by centrifugation,then fixed with PBS containing 1% paraformaldehyde at room temperaturefor 5 minutes, followed by washing with PBS six times by centrifugation.700 μl of 1×10⁶/ml mouse spleen cells and 350 μl of 4×10⁴/ml ST2 cellswhich were suspended in α-MEM culture medium containing 10⁻⁸ Mactive-form vitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovineserum, were added to a 24-well plate. TC insert (Nunc Co.) was set ineach well. The above-described fixed COS cells (350 μl) diluted tovarious concentrations with the above-mentioned culture medium and OCIFsolution (50 μl) were added to the TC insert and cultured for 6 days at37° C. As a result, it was confirmed that the osteoclasts formationinhibitive activity of OCIF can be inhibited by the protein encoded bythe cDNA of the present invention.

Example 14 Expression of Secreted-Form OBM (1) Construction of a Plasmidfor the Expression of Secreted-Form OBM

A PCR reaction was carried out using OBM HF (SEQ ID NO:7) and OBM XR(SEQ ID NO:8) as primers and pOBM291 as a template. After purificationby agarose gel electrophoresis, the product was digested withrestriction enzymes HindIII and EcoRI, and further purified by agarosegel electrophoresis. The purified fragment (0.6 kb), Hind III/EcoRIfragment (5.2 kb) of pSec TagA (Invitrogen Co.), and EcoRI/PmacIfragment (0.32 kb) of OBM cDNA were ligated using a a ligation kit ver.2 (Takara Shuzo Co.) Escherichia coli DH5α was transformed using thereaction product. Plasmids were purified by means of alkali SDS methodfrom the resulting ampicillin resistant strains and digested withrestriction enzymes to select a plasmid with fragments of a length of0.6 Kb and 0.32 kb being inserted into pSec TagA. Selected plasmid wasidentified as having a sequence encoding the secreted-form OBM(nucleotide sequence: 338-1355 in SEQ ID NO:2, amino acid sequence:72-316 in the SEQ ID NO:1) by sequencing using a dye terminator cyclesequencing FS kit (Perkin Elmer Co.). This plasmid was digested withrestriction enzymes Nhel and Xhol to isolate a fragment (1.0 kb)containing the secreted-form OBM cDNA by agarose gel electrophoresis.This fragment was inserted into the NheI/XhoI fragment (10.4 kb) of anexpression vector, pCEP4 (Invitrogen Co.), using a ligation kit andEscherichia coli DH5α was transformed using the reaction productthereof. Plasmids were purified by alkali SDS method from the resultingampicillin resistant strains and digested with restriction enzymes toselect an Escherichia coli strain having the secreted-form OBMexpression plasmid (pCEPsOBM) with the correct structure. TheEscherichia coli strain containing the pCEP sOBM was cultured and pCEPsOBM was purified using QIA filter plasmid midi kit (QIAGEN Co.).

(2) Expression of Secreted-Form OBM

293-EBNA cells were suspended in IMDM containing 10% FCS (IMDM-10% FCS)and seeded into a 24-well plate coated with collagen (manufactured bySumitomo Bakelite Co., Ltd.) in a cell density of 2×10⁵/2 ml/well andcultured overnight. The cells were transfected with 1 μg of pCEP sOBM orpCEP4 using 4 μl of Lipofectamine (Gibco Co.). After culturing for twodays in 0.5 ml of a serum-free IMDM or IMDM-10% FCS, the conditionedmedium was collected. Expression of the secreted-form OBM in theconditioned medium was confirmed as follows. Sodium hydrogen carbonatewas added to the conditioned medium to a final concentration of 0.1 Mand the solution was added to a 96-well plate. The plate was allowed tostand overnight at 4° C., thereby immobilizing OBM in the conditionedmedium on the 96-well plate. The plate was filled with a Block Ace™(Snow Brand Milk Products Co., Ltd.) solution diluted four-fold with PBS(B—PBS) and allowed to stand for two hours at room temperature to blockresidual binding sites of the plate. After the addition to each well of100 μl of 3-100 ng/ml of OCIF which was diluted with B—PBS, the platewas allowed to stand for two hours at 37° C., followed by washing withPBS containing 0.05% Tween 20(PBS-T). Then, 100 μl of aperoxidase-labeled rabbit anti-OCIF polyclonal antibody described in WO96/26217 which was diluted with B—PBS was added to each well. Afterallowing to stand for two hours at 37° C., the wells were washed sixtimes with PBS-T Then, a TMB solution (TMB Soluble Reagent, HighSensitivity, Scytek Co.) was added in the amount of 100 μl per well andallowed to stand at room temperature for about 10 minutes, whereupon thereaction was terminated by the addition of 100 μl of a terminationsolution (Stopping Reagent, Scytek Co.) to each well. Absorbance at 450nm of each well was measured by a microplate reader. The results areshown in FIG. 12 which indicates that the absorbance at 450 nm increasedaccording to the concentration of the added OCIF in the plate in whichthe conditioned medium of the cells transfected with pCEP sOBM wasimmobilized. On the other hand, no increase in absorbance was seen inthe plate in which the conditioned medium of the cells transfected withvector pCEP4 was immobilized. FIG. 13 shows the results of an experimentwherein the proportion of the conditioned medium which is used forimmobilization was changed within a range of 5-90% and a specificconcentration of OCIF (50 ng/ml) was added. It can be seen that theabsorbance at 450 nm increased according to the increase in theproportion of the conditioned medium in the plate wherein theconditioned medium of the cells transfected with pCEP sOBM wasimmobilized, whereas no such increase in absorbance was seen in theplate wherein the conditioned medium of the cells transfected withvector pCEP4 was immobilized. From these results, it was confirmed thatsecreted-form OBM is produced into the conditioned medium of the cellstransfected with pCBP sOBM.

Example 15 Expression of Thioredoxin-OBM Fusion Protein (Trx-OBM) (1)Construction of a Thioredoxin-OBM Fusion Protein (Trx-OBM) ExpressionVector

Ten μl of 10×ExTaq buffer (Takara Shuzo Co.), 8 μl of 10 mM dNTP (TakaraShuzo Co.), 77.5 μl of sterilized distilled water, 2 μl of an aqueoussolution of pOBM291 (10 ng/μl), 1 μl of primer OBM3 (100 μmol/μ1, SEQ IDNO:9), 1 μl of primer OBMSalR2 (100 μmol/μl, SEQ ID NO:10), and 0.5 μlof ExTaq (5 u/μl) (Takara Shuzo Co.) were mixed and reacted (PCRreaction) in a micro centrifuge tube. After reacting at 95° C. for 5minutes, at 50° C. for one second, at 55° C. for one minute, at 74° C.for one second, and at 72° C. for 5 minutes, a cycle reaction consistingof a reaction at 96° C. for one minute, at 50° C. for one second, at 55°C. for one minute, at 74° C. for one second, and at 72° C. for 3minutes, was repeated 25 times. From the total reaction liquid DNAfragment of about 750 bp was purified by 1% agarose gel electrophoresisusing a QIAEX II gel extraction kit (QIAGEN Co.). The whole amount ofpurified DNA fragment was digested with restriction enzymes SaII andEcoRI (Takara Shuzo Co.) and subjected to an 1.5% agarose gelelectrophoresis to purify a DNA fragment of about 160 bp (Fragment 1),which was dissolved in 20 μl of sterilized distilled water. In the samemanner, a DNA fragment of about 580 bp (Fragment 2) obtained bydigesting 4 μg of pOBM291 with restriction enzymes BamH1 and EcoRI(Takara Shuzo Co.) and a DNA fragment of about 3.6 kb (Fragment 3)obtained by digesting 2 μg of pTrXFus (Invitrogen Co.) with restrictionenzymes BamHI and SaII (Takara Shuzo Co.) were respectively purified anddissolved in 20 μl of sterilized distilled water. The QIAEXII gelextraction kit was used for the purification of DNA fragments. Fragments1-3 were ligated by incubating at 16° C. for 2.5 hours using DNAligation kit ver.2 (Takara Shuzo Co.). Using the ligation reactionliquid, Escherichia coli strain GI724 (Invitrogen Co.) was transformedaccording to the method described in the Instruction Manual ofThioFusion Expression System (Invitrogen Co.). A microorganism strainwith plasmid in which the OBM cDNA fragment (nucleotide sequence:350-1111 in the SEQ ID NO:2, amino acid sequence: 76-316 in the SEQ IDNO:1) is fused in frame to a thioredoxin gene was selected from theresulting ampicillin resistant transformants by the analysis ofrestriction maps obtained by digestion with restriction enzymes and DNAsequence determination. The microorganism strain thus obtained was namedGI724/pTrxOBM25.

(2) Expression of OBM in Escherichia coli

GI724/pTrxOBM25 and GI724 containing pTrxFus (GI724/pTrxFus) wererespectively cultured six hours with shaking at 30° C. in 2 ml ofRMG-Amp culture medium (0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.05% NaCl, 0.1%NH₄Cl, 1.2% casamino acid (Difco Co.), 1% glycerol, 1 mM MgCl₂, and 100μg/ml ampicillin (Sigma Co.), pH 7.4). The broth 0.5 ml of the broth wasadded to 50 ml of Induction culture medium (0.6% Na₂HPO₄, 0.3% KH₂PO₄,0.05% NaCl, 0.1% NH₄Cl, 0.2% casamino acid, 0.5% glucose, 1 mM MgCl₂,100 μg/ml ampicillin, pH 7.4) and cultured with shaking at 30° C. WhenOD₆₀₀ nm reached about 0.5, L-tryptophan was added to a finalconcentration of 0.1 mg/ml, followed by shaking the culture at 30° C.for an additional 6 hours. The culture broth was centrifuged at 3000×gto collect the cells, which were suspended in 12.5 ml of PBS (10 mMphosphate buffer, 0.15 M NaCl, pH 7.4). The suspension was subjected toan ultrasonic generator (Ultrasonics Co.) to disrupt the cells. Thedisrupted cells were centrifuged at 7000×g for 30 minutes to obtain asupernatant liquid as a soluble protein fraction. Ten μl of this solubleprotein fraction was subjected to SDS polyacrylamide (10%)electrophoresis under reducing conditions. As a result, a band with amolecular weight of 40 kDa which was not detected in the soluble proteinfraction of GI724/pTrxFus was found in the soluble protein fraction ofGI724/pTrxOBM25 (FIG. 14). Accordingly, it was confirmed that a fusionprotein (Trx-OBM) of thioredoxin and OBM was expressed in Escherichiacoli.

(3) Binding Capability of Trx-OBM to OCIF

Binding of the expressed Trx-OBM to OCIF was confirmed according to thefollowing experiment. Anti-thioredoxin antibody (Invitrogen Co.) whichwas diluted to 5000-fold with 10 mM sodium hydrogen carbonate solutionwas added to a 96-well immunoplate (Nunc Co.) in the amount of 100 μlper well. After being allowed to stand overnight at 4° C., the liquid inthe wells was discarded. Two hundred μl of a solution prepared bydiluting Block Ace™ (Snow Brand Milk Products Co., Ltd.) two-fold withPBS (BA-PBS) was added to each well. After being allowed to stand forone hour at room temperature, the solution was discarded and solubleprotein fractions originating from the above-described GI724/pTrxOBM25or GI724/pTrxFus, each diluted with BA-PBS in various concentrationswere added to each well in the amount of 100 μl. After being allowed tostand for two hours at room temperature, each well was washed threetimes with PBS-T and charged with 100 μl of OCIF (100 ng/ml) which wasdiluted with BA-PBS. After being allowed to stand for two hours at roomtemperature, each well was washed three times with PBS-T and chargedwith 100 μl of peroxidase-labeled rabbit anti-OCIF polyclonal antibody(described in WO 96/26217) which was diluted 2,000-fold with BA-PBS.After being allowed to stand for two hours at room temperature, eachwell was washed six times with PBS-T and charged with 100 μl of TMBsolution (TMB Soluble Reagent, High Sensitivity, Scytek Co.). Afterbeing allowed to stand for about 10 minutes at room temperature, eachwell was charged with 100 μl of termination solution (Stopping Reagent,Scytek Co.). Absorbance of each well at 450 nm was measured by amicroplate reader. The results are shown in FIG. 15. There was nodifference in absorbance between the sample with the soluble proteinfraction originating from GI724/pTrxFus added thereto and the samplewithout the addition of this soluble protein fraction. On the otherhand, the absorbance increased in the samples to which the solubleprotein fraction originating from GI724/pTrxOBM25 was added inproportion to the concentration of the soluble protein fraction. Theresults of the other experiment wherein the dilution rate of the solubleprotein fraction was maintained constant (1%) while adding OCIF dilutedwith BA-PBS in different concentrations (0-100 ng/ml) are shown in FIG.16. It can be seen that the absorbance remained low at anyconcentrations of OCIF in samples using a soluble protein fractionoriginating from GI724/pTrxFus, whereas the absorbance increased inproportion to the OCIF concentration in the samples to which the solubleprotein fraction originating from GI724/pTrxOBM25 was added. Based onthese results, it was confirmed that Trx-OBM which is produced fromGI724/pTrxOBM25 has a capability of binding to OCIF.

(4) Large-Scale Cultivation of Escherichia coli which Produces Trx-OBM

GI724/pTrxOBM25 cells were spread on RMG-Amp agar (0.6% Na₂PO₄, 0.3%KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 2% casamino acid, 1% glycerol, 1 mMMgCl₂, 100 μg/ml ampicillin, 1.5% agar, pH 7.4) using a platinumtransfer loop. The cells were cultured overnight at 30° C. The culturedcells were suspended in 10 ml of Induction medium. The suspension wasadded 5 ml for each to two 2 1 Erlenmeyer flasks containing 500 ml ofInduction medium and cultured at 30° C. with shaking. When the OD₆₀₀ nmreached about 0.5, L-tryptophan was added to a final concentration of0.1 mg/ml. Culturing with shaking was continued for six hours at 30° C.The culture broth was centrifuged for 20 minutes at 3000×g to collectthe cells, which were suspended in 160 ml of PBS. The suspension wassubjected to an ultrasonic generator (Ultrasonics Co.) to disrupt thecells. The supernatant liquid was centrifuged for 30 minutes at 7000×gto obtain a soluble protein fraction.

(5) Preparation of OCIF-Immobilized Affinity Column

Two g of TSKgel AF-Tolesyl Toyopal 650 (Tosoh Corp.) and 40 ml of 1.0 Mpotassium phosphate buffer (pH 7.5) containing 35.0 mg of recombinantOCIF, which was prepared according to the method described in WO96/26217, were mixed. The mixture was gently shaken overnight at 4° C.to effect a coupling reaction. The reaction mixture was centrifuged toremove the supernatant. To inactivate excess active residues, 40 ml of0.1 M Tris-HCl buffer (pH 7.5) was added to the precipitated carrier andthe mixture was gently shaken at room temperature for one hour. Thecarrier in a column was washed with 0.1 M glycine-HCl buffer (pH 3.3)containing 0.01% Polysorbate 80 and 0.2 M NaCl and 0.1 M sodium citratebuffer (pH 2.0) containing 0.01% Polysorbate 80 and 0.2 M NaCl. Thecarrier in the column was equilibrated by charging twice with 10 mMsodium phosphate buffer (pH 7.4) containing 0.01% Polysorbate 80.

(6) Purification of Trx-OBM Using OCIF-Immobilized Affinity Column

Unless otherwise indicated, purification of Trx-OBM was carried out at4° C. The above-mentioned OCIF-immobilized affinity carrier (10 ml) andthe soluble protein fraction (120 ml) prepared in Example 15(4) weremixed. The mixture was gently shaken overnight at 4° C. in four 50-mlcentrifuge tubes using a rotor. An Econo-column™ (internal diameter: 1.5cm, length: 15 cm, manufactured by BioRad Co.) was filled with thecarrier in the mixture. The column was charged with 300 ml of PBScontaining 0.01% Polysorbate 80, 100 ml of 10 mM sodium phosphate buffer(pH 7.0) containing 0.01% Polysorbate 80 and 2 M NaCl, and 100 ml of 0.1M glycine-HCl buffer (pH 3.3) containing 0.01% Polysorbate 80 and 0.2 MNaCl, in that order. Next, proteins adsorbed in the column were elutedwith 0.1 M sodium citrate buffer (pH 2.0) containing 0.01% Polysorbate80 and 0.2 M NaCl. The eluate was collected in 5 ml portions. Eachfraction thus collected was immediately neutralized with addition 10%volume of 2 M Tris buffer (pH 8.0). Presence or absence of Trx-OBM inthe eluted fractions was determined according to the method previouslydescribed in Example 15(3) (the binding capability to OCIF). Thefractions containing Trx-OBM were collected and purified further.

(7) Purification of Trx-OBM by Gel Filtration

About 25 ml of Trx-OBM fractions obtained in Example 15(6) wasconcentrated to about 0.5 ml by centrifuge using Centriplus 10 andCentricon 10 (Amicon Co.). This sample was applied to a Superose 12 HR10/30 column (1.0×30 cm, Pharmacia Co.) previously equilibrated with PBScontaining 0.01% Polysorbate 80. For the separation, PBS containing0.01% Polysorbate 80 was used as a mobile phase at a flow rate of 0.25ml/min. The eluate from the column was collected in 0.25 ml portions.The Trx-OBM in the thus-collected fractions was detected by the samemethod as previously described in Example 15(3) and bySDS-polyacrylamide electrophoresis (10-15% polyacrylamide gel, PharmaciaCo.) using Phast System (Pharmacia Co.) and silver staining. Fractions(Fr. 20-23) containing purified Trx-OBM were collected and the proteinconcentration of Trx-OBM was determined. The measurement of the proteinconcentration was carried out using bovine serum albumin as a standardsubstance using DC-Protein assay kit (BioRad Co.).

Example 16 Osteoclast Formation-Inducing Activity of OBM

pOBM291 and pcDL-SR α296 were respectively transfected into COS-7 cellsusing Lipofectamine (Gibco Co.). The cells were cultured in DMEMcontaining 10% FCS for one day, trypsinized, plated on cover slips (15mm round shape, manufactured by Matsunami Co.) in 24-well plates at5×10⁴ cells per well, and cultured for 2 days. The culture plate waswashed once with PBS. The cells were fixed with PBS containing 1%paraformaldehyde at room temperature for 8 minutes. The plate on whichthe fixed cells were attached was washed 6 times with PBS, then 700 μlof mouse spleen cells suspended at 1×10⁶/ml in α-MEM containing 10⁻⁸ Mactive-form vitamin D₃, 10⁻⁷ dexamethasone, and 10% fetal bovine serumwere added to each well. Millicell PCF (Millipore Co.) was set in eachwell and a suspension of ST2 cells in the above-mentioned culture medium(4×10⁴/ml) were added, 700 μl per well, into the Millicell PCF followedby incubation at 37° C. for 6 days. After the culture, the Millicell PCFwas removed, the plate was washed once with PBS, and the cells werefixed with acetone-ethanol solution (50:50) for one minute. Then, cellsexhibiting tartaric acid-resistant acid phosphatase activity (TRAP),which is a specific marker for osteoclast, were selectively stainedusing LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). As a result ofmicroscopic observation, TRAP-positive cells were not detected in thewells in which COS-7 cells transfected with pcDL-SR α296 were fixed. Incontrast, 45±18 (average±standard deviation, n=3) TRAP positive cellswere observed in the wells in which COS-7 cells transfected with pOBM291were fixed. Moreover, it was also confirmed that calcitonin bound tothese TRAP positive cells. Based on these findings, it has been proventhat OBM has osteoclast formation-inducing activity.

Example 17 Osteoclast Formation-Inducing Activity of Trx-OBM andSecreted-Form OBM

Mouse spleen cells were suspended in α-MEM containing 10⁻⁸ M active-formvitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovine serum at aconcentration of 2×10⁶/ml. The suspension was added to a 24 well platein the amount of 350 μl per well. Each well was then charged with 350 μlof a solution prepared by diluting purified Trx-OBM with theabove-mentioned culture medium (40 ng/ml), 350 μl of solution preparedby 10-fold diluting conditioned medium which was produced by culturing293-EBNA cells, in which pCEP sOBM or pCEP4 were transfected, inIMDM-10% FCS, with the above-mentioned culture medium, or 350 μl only ofthe above-mentioned culture medium. The Millicell PCF (Mollipore Co.)was set on each well, to which 600 μl of ST2 cells which was suspendedin the above-mentioned culture medium (4×10⁴/ml) were added. Aftercultured for six days, Millicell PCF was removed. The plate was washedonce with PBS and the cells were fixed with acetone-ethanol solution(50:50) for one minute. Then, the cells exhibiting the tartaric acidresistant acidic phosphatase activity (TRAP activity) were selectivelystained using LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). The result ofmicroscopic observation revealed that no cells exhibiting the TRAPactivity were detected in the wells to which no Trx-OBM was added,whereas 106±21 (average±standard deviation, n=3) TRAP-positive cellswere observed in the wells to which Trx-OBM was added. Similarly, whileno cells exhibiting TRAP activity were detected in the wells to whichconditioned medium of 293-EBNA transfected with pCEP4 had been added,120±31 (average ±standard deviation, n=3) TRAP positive cells wereobserved in the wells to which conditioned medium of 293-EBNAtransfected with pCEP sOBM had been added. Moreover, it was alsoconfirmed that calcitonin binds to these TRAP positive cells. Theseresults have proven that Trx-OBM and secreted-form OBM exhibitosteoclast formation-inducing activity.

Example 18 Identity of the Protein OBM Expressed by the cDNA of thePresent Invention and the Natural Type OCIF-Binding Protein of thePresent Invention (1) Preparation of Rabbit Anti-OBM Polyclonal Antibody

Three male Japanese white rabbits (weight: 2.5-3.0 kg, supplied byKitayama Labes Co.) were immunized with the purified OBM(thioredoxin-OBM fusion protein) produced according to the method inExamples 14(6) and 14(7) by subcutaneously injecting 1 ml/dose ofemulsion prepared by mixing 200 μg/ml of the purified OBM with equalvolume of Freund's complete adjuvant (DIFCO Co.), six times, once aweek. Ten days after the last immunization, the rabbits wereexsanguinated. Antibody was purified from the serum as follows. Ammoniumsulfate was added to the antiserum which was diluted two-fold with PBSto a final concentration of 40% (w/v %). After being allowed to standfor one hour at 4° C., the mixture was centrifuged for 20 minutes at8000×g to obtain a precipitate. The precipitate was dissolved in a smallamount of PBS, dialyzed against PBS at 4° C., and loaded to a ProteinG-Sepharose column (manufactured by Pharmacia Co.) After washed withPBS, the adsorbed immunoglobulin G was eluted with 0.1 M glycine-HClbuffer solution (pH 3.0). The eluate was immediately neutralized with1.5 M Tris-HCl buffer (pH 8.7). After dialyzing the eluted proteinfractions against PBS, the absorbance at 280 nm was measured todetermine the protein concentration (E^(1%) 13.5). Anti-OBM antibodylabeled with horseradish peroxidase was prepared using amaleimide-activated peroxidase kit (Pierce Co.) as follows. 80 μg ofN-succinimide-5-acetyl thioacetic acid was added to 1 mg of the purifiedantibody and reacted at room temperature for 30 minutes. Five mg ofhydroxylamine was added to the resulting mixture to deacetylate theantibody. The modified antibody was fractionated using a polyacrylamidedesalting column. The protein fractions were mixed with 1 mg ofmaleimide-activated peroxidase and reacted for one hour at roomtemperature to obtain enzyme-labeled antibody.

(2) Capability of Rabbit Anti-OBM Polyclonal Antibody to InhibitSpecific Binding of the Protein (OBM) Expressed by the cDNA of thePresent Invention or the Natural Type Protein of the Present Inventionwith OCIF

Purified OBM (thioredoxin-OBM fused protein) obtained according to themethod described in the Examples 15(6) and 15(7) and the natural typepurified OCIF-binding protein of the Example 2(4) were dissolvedrespectively in 0.1 M sodium carbonate buffer to a concentration of 2μg/ml. An aliquot of each solution was added 100 μl per wellrespectively to a 96-well immunoplate (manufactured by Nunc Co.). Theplate was allowed to stand overnight at 4° C. 200 μl of 50% Block Acewas added to each well and the plate was allowed to stand at roomtemperature for one hour. After washing each well three times with PBScontaining 0.1% Polysorbate 20 (P20-PBS), 100 μl of rabbit anti-OBMantibody solution which was dissolved in 25% Block Ace prepared withP20-PBS to a concentration of 200 μg/ml or 100 μl of 25% Block Ace(containing no antibody) was added to each well, followed by incubationat 37° C. for one hour. Each well was washed three times with P20-PBSand charged with 100 μl/well of a binding test solution (P20-PBScontaining 0.2% calf serum albumin, 20 mM Hepes, and 0.1 mg/ml heparin)to which 20 ng/ml of ¹²⁵I-labeled OCIF described in the Example 8(3) wasadded. Alternatively, each well was charged with 100 μl/well of anotherbinding test solution containing 8 μg/ml of unlabeled OCIF in additionto 20 ng/ml ¹²⁵I-labeled OCIF. After incubating these immunoplates at37° C. for one hour, the wells were washed with P20-PBS six times. Theamount of ¹²⁵I in each well was measured by a gamma counter. The resultsare shown in FIG. 17. As shown in the figure, both the purified OBMexpressed using the cDNA of the present invention and the protein thatspecifically bind the natural type OCIF-specifically binding protein ofthe present invention do not bind to the ¹²⁵I-labeled OCIF at all, whenthey were treated with the rabbit anti-OBM polyclonal antibody, whereasboth proteins bound ¹²⁵I-labeled OCIF when untreated with the antibody.The binding of both proteins to ¹²⁵I-labeled OCIF was confirmed to beclearly specific, because those bindings are significantly inhibited bythe addition of 400-fold concentration of unlabelled OCIF (8 μg/ml).Based on the results described above, the rabbit anti-OBM polyclonalantibody recognizes both the OBM which is the protein expressed usingthe cDNA of the present invention and the natural-type OCIF-bindingprotein of the present invention, and it inhibits the specific bindingof these proteins with OCIF.

Example 19 Cloning of Human OBM cDNA (1) Preparation of Mouse OBM Primer

The mouse OBM primers prepared according to the method of the Examples(OBM#3 and OBM#8) described above, were used for screening of human OBMcDNA. The sequences are shown in the Sequence Table, SEQ ID NO:9 and SEQID NO:6, respectively.

(2) Isolation of Human OBM cDNA Fragment by PCR

Human OBM cDNA fragments were obtained by PCR using the mouse OBM cDNAprimers prepared in (1) above and Human Lymph Node Marathon ready cDNA(Clontech Co.) as a template. The conditions for PCR were shown asfollows:

10 × EX Taq buffer (Takara Shuzo Co.) 2 μl 2.5 mM dNTP 1.6 μl cDNAsolution 1 μl EX Taq (Takara Shuzo Co.) 0.2 μl Distilled water 14.8 μl40 μM primer OBM#3 0.2 μl 40 μM primer OBM#8 0.2 μl

These solutions were mixed in a microfuge tube and pre-incubated at 95°C. for 2 minutes, followed by 40 cycles of a three-stage reactionconsisted of reactions at 95° C. for 30 seconds, at 57° C. for 30seconds, and at 72° C. for 2.5 minutes. After the reaction, the solutionwas incubated for 5 minutes at 72° C. and a portion of the solution wassubjected to electrophoresis on an agarose gel. A DNA fragment (about690 bp) amplified by the mouse OBM cDNA primers described above wasdetected.

(3) Purification of the Human OBM cDNA Fragment Amplified by PCR andDetermination of the Nucleotide Sequence

The human OBM cDNA fragment obtained in Example 19(2) was separated byelectrophoresis on an agarose gel and further purified using a QIAEX gelextraction kit (Qiagen Co.). PCR was again performed using the purifiedhuman OBM cDNA fragment as a template and the mouse OBM cDNA primersdescribed above, to produce a large quantity of the human OBM cDNAfragment. The DNA fragment was purified by a QIAEX gel extraction kit inthe same manner as above. The nucleotide sequence of the purified humanOBM cDNA fragment was determined using a Taq Dye Deoxy Terminator CycleSequencing FS kit (Perkin Elmer Co.) using OBM#3 or OBM#8 (SEQ ID NO:9and SEQ ID NO:6 respectively) as a primer. When compared with thesequence of corresponding area of the mouse OBM cDNA, the nucleotidesequence of the human OBM cDNA fragment showed 80.7% homology with thatof the mouse OBM cDNA.

(4) Screening of a Full-Length Human OBM cDNA by Hybridization Using theHuman OBM cDNA Fragment (about 690 bp) as a Probe

A full-length human OBM cDNA was screened using the human OBM cDNAfragment (about 690 bp) that was purified in the Example 19(3) andlabeled with [α³²P] dCTP using a Megaprime DNA Labeling kit (AmershamCo.). Human Lymph Node 5′-STRETCH PLUS cDNA library (Clontech Co., theU.S.A) was screened using the DNA probe. According to the manufacturer'sprotocol, Escherichia coli C600 Hfl was infected with the recombinantphage for 15 minutes at 37° C. The infected Escherichia coli was addedto an LB agar (1% tryptone, 0.5% yeast extract, 1% NaCl, 0.7% agar)which was heated at 45° C. The LB agar was poured onto an LB agar platecontaining 1.5% agar. After overnight incubation at 37° C., HyBond-N™(Amersham Co.) was placed to the plate on which plaques were producedand stored for about 3 minutes. According to a conventional method, thefilter was treated with alkaline solution, neutralized, and dipped in2×SSC solution. DNA was then immobilized onto the filter using the UVCROSSLINKER (Stratagene Co.). The resulting filter was dipped intoRapid-hyb buffer (Amersham Co.). After pretreatment for 15 minutes at65° C., the filter was placed in Rapid-hyb buffer containing theheat-denatured human OBM cDNA fragment (about 690 bp, 5×10⁵ cpm/ml)described above. After overnight hybridization at 65° C., the filter waswashed with 2×SSC, 1×SSC, and 0.1×SSC, each containing 0.1% SDS, in thisorder respectively for 15 minutes at 65° C. Several positive clonesobtained were further purified by repeating the screening twice. A clonepossessing an insert (about 2.2 kb) was selected from the purifiedclones and was used in the following experiments. This purified phagewas named λhOBM. About 10 μg of DNA was obtained from the purified λhOBMusing a QIAGEN Lambda kit (Qiagen Co.) according to the manufacturer'sprotocol. The DNA was digested with restriction enzyme SalI andsubjected to electrophoresis on an agarose gel to separate the hOBMinsert cDNA (about 2.2 kb). This DNA fragment purified using the QIAEXgel extraction kit (Qiagen Co.) was digested with restriction enzymeSaII and inserted into plasmid pUC19 (MBI Co.) which was previouslydigested with a restriction enzyme SaII and dephosphorylated, using aDNA ligation kit ver. 2 (Takara Shuzo Co.). Escherichia coli DH 5α(Gibco BRL Co.) was transformed with the pUC19 containing the resultingDNA fragment. The resulting transformant was named pUC19hOBM. Thetransformant was grown and pUC19hOBM in which the human OBM cDNA (about2.2 kb) was inserted and purified by a conventional method.

(5) Determination of Nucleotide Sequence of cDNA Encoding the EntireAmino Acid Sequence of Human OBM

The nucleotide sequence of the resulting human OBM cDNA obtained inExample 19(4) was determined using the Taq Dye Deoxy Terminator CycleSequencing FS kit (Parkin Elmer Co.). Specifically, the nucleotidesequence of the inserted fragment was determined using pUC19hOBM as atemplate. As primers, primers for the determination of the nucleotidesequence of the inserted fragment DNA in pUC19hOBM, M13 Primer M3 andM13 Primer RV (manufactured by Takara Shuzo Co.), and a syntheticprimer, human OBM#8, designed based on the nucleotide sequence of thehuman OBM cDNA fragment (about 690 bp) were used.

The nucleotide sequence of the primers used, M13 Primer M3 and M13Primer RV, are respectively shown as the Sequence ID No. 4 and No. 5.The amino acid sequence of human OBM deduced from the nucleotidesequence of human OBM cDNA is shown in the Sequence Table as Sequence IDNo. 11. The nucleotide sequence of human OBM cDNA is shown as SequenceID No. 12.

The Escherichia coli which was transformed by the pUC19hOBM, which isthe plasmid containing the resulting human OBM cDNA, was deposited inNational Institute of Bioscience and Human Technology, Agency ofIndustrial Science and Technology, on Aug. 13, 1997 as deposition No.FERM BP-6058.

Example 20

Radioiodination of OCIF with ¹²⁵I and Quantitative Analysis of¹²⁵I-Labled OCIF by ELISA

OCIF was labeled with ¹²⁵I using the IODO-GEN method. Twenty μl of 2.5mg/ml IODO-GEN-chloroform solution was transferred to a 1.5 ml Eppendorftube and the chloroform was evaporated at 40° C., thereby providing atube coated with IODO-GEN. The tube was washed three times with 400 μlof 0.5 M sodium phosphate buffer solution (Na-Pi, pH 7.0), followed bythe addition of 5 μl of 0.5 M Na-Pi (pH 7.0). To this tube was added 1.3μl (18.5 MBq) of Na—¹²⁵I solution (Amersham Co., NEZ-033H), immediatelyfollowed by the addition of 10 μl of 1 mg/ml OCIF solution (monomer typeor dimer type). The mixture was mixed in a vortex mixer and allowed tostand at room temperature for 30 seconds. This solution was transferredto a tube to which 80 μl of 0.5 M Na-Pi (pH 7.0) solution containing 10mg/ml potassium iodine and 5 μl of a phosphate buffered saline solutioncontaining 5% bovine serum albumin (BSA-PBS) were previously added. Thesolution was mixed, applied to a spin column (1 ml, G-25 Sephadex fine,manufactured by Pharmacia Co.) which was equilibrated with BSA-PBS inadvance, and centrifuged for 5 minutes at 2,000 rpm. Four hundred μl ofBSA-PBS was added to the fractions eluted from the column. After mixing,2 μl of the solution was used to measure the radioactivity by a gammacounter. The radiochemical purity of the ¹²⁵I-labeled OCIF solutionobtained above was measured by counting the radioactivity of fractionsprecipitated by 10% trichloroacetic acid (TCA).

The biological activity of the ¹²⁵I-labeled OCIF was measured accordingto the method described in WO 96/26217. The concentration of the¹²⁵I-labeled OCIF was measured using the ELISA method as follows.Specifically, 50 mM NaHCO₃ (pH 9.6) in which rabbit anti-OCIF polyclonalantibody described in the WO 96/26217 was dissolved to a concentrationof 2 μg/ml was added to each well of a 96-well immunoplate (MaxiSorp™,manufactured by Nunc Co.) in the amount of 100 μl per well. After thesewells were allowed to stand overnight at 4° C., solution was removed.Then the wells were charged with a mixed aqueous solution of Block Ace™(Snow Brand Milk Products Co., Ltd.) and a phosphate buffered salinesolution (25:75) (B—PBS) in the amount of 200 μl/well. The plate wasthen allowed to stand for two hours at room temperature. After thesolution was removed, the wells were washed three times with a phosphatebuffered saline solution containing 0.01% Polysolvate 80 (P—PBS). Next,B—PBS containing ¹²⁵I-labeled OCIF sample or the standard OCIF was addedin the amount of 100 μl/well. The plate was then allowed to stand fortwo hours at room temperature. After the solution was removed, each wellwas washed six times with 200 μl of P—PBS. A solution prepared bydiluting peroxidase-labeled rabbit anti-OCIF polyclonal antibody withB—PBS was added in the amount of 100 μl/well. The plate was allowed tostand for two hours at room temperature. After the solution was removed,the wells were washed six times with 200 μl of P—PBS. Then, a TMBsolution (TMB Soluble Reagent, High Sensitivity, Scytek Co.) was addedin the amount of 100 μl/well. After being allowed to stand at roomtemperature for 2-3 minutes, 100 μl of a termination solution (StoppingReagent, Scytek Co.) was added to each well. Absorbance of each well wasmeasured at 450 nm using a microplate reader. The concentration of¹²⁵I-labeled OCIF was determined with a calibration curve prepared usingthe standard OCIF.

Example 21 Expression of the Protein Encoded by cDNA of the PresentInvention

(1) Construction of hOBM Expression Vector for Animal Cells

pUChOBM was digested with restriction enzyme SaII and a DNA fragment(about 2.2 kb) were purified by electrophoresis on an 1% agarose gel.The ends of the DNA fragments were blunted using a DNA blunting kit(Takara Shuzo Co.) (blunted hOBM cDNA fragment). Expression plasmidpcDL-SRα296 (Molecular and Cellar Biology, Vol. 8, pp 466-472 (1988))was digested with restriction enzyme EcoRI, blunted with blunting kitand ligated with the blunted hOBM cDNA fragment using DNA ligation kitver. 2. Escherichia coli DHα was transformed with the ligation reaction.A plasmid in the resulting ampicillin resistant transformant wassubjected to digestion with restriction enzyme to analyze the DNArestriction map and determine the DNA sequence. As a result, a strainhaving a plasmid in which hOBM cDNA is inserted in the same direction oftranscription as that of SRα promotor was selected. The microorganismstrain was named DH5 α/phOBM.

(2) Expression of Human OBM in COS-7 Cells

Escherichia coli DH5α/phOBM was cultured and plasmid phOBM was purifiedusing Qiafilter Plasmid Midi kit (Qiagen Co.) phOBM was transfectedusing Lipofectamine into COS-7 cells in the wells of a 6-well plate andcultured for two days in DMEM containing 10% fetal bovine serum. Theculture medium was replaced with cysteine-methionine-free DMEM(manufactured by Dainippon Seiyaku Co., Ltd.) to which 5% dialysed fetalbovine serum was added (88 μl/well). The cells were incubated for 15minutes, followed by addition of 14 μl of Express Protein LabelingMix(NEN Co., 10 mCi/ml). After four hours incubation, 200 μl of DMEMcontaining 10% fetal bovine serum was added to each well. The cells werecultured for one hour and washed twice with PBS. Then, 0.5 ml of a TSAbuffer (10 mM Tris-HCl containing 0.14 M NaCl and 0.025% NaN₃, pH 8.0)containing 1% Triton X-100, 1% bovine hemoglobin, 10 μg/ml leupeptin,0.2 TIU/ml aprotinin, and 1 mM PMSF was added to each well and themixtures were allowed to stand for one hour on ice. The cells were mixedby pipetting and centrifuged at 3,000×g, for 10 minutes at 4° C., toobtain supernatants. Two hundred μl of a dilution buffer (TSA buffercontaining 0.1% Triton X-100, 0.1% bovine hemoglobin, 10 μg/mlleupeptin, 0.2 TIU/ml aprotinin, and 1 mM PMSF) was added to 100 μl ofthe supernatant from each well. The resulting mixtures were agitated at4° C. for one hour together with Protein A Sepharose (50 μl) andcentrifuged at 1,500×g for one minute at 4° C., to collect supernatants,thereby removing the protein which non-specifically adsorbed Protein ASepharose. OCIF (1 μg) was added to the supernatants and the mixtureswere agitated for one hour at 4° C. to bind human OBM and OCIF. Then,rabbit anti-OCIF polyclonal antibody (50 μg) was added, followed byagitation at 4° C. for one hour. Protein A Sepharose (10 μl) was addedto the resulting solution, followed by agitation at 4° C. for anadditional hour. The mixtures thus obtained were centrifuged for 1minute at 1,500×g at 4° C. to collect precipitates. The precipitateswere washed twice with a dilution buffer, twice with bovinehemoglobin-free dilution buffer, once with TSA buffer, and once with 50mM Tris-HCl (pH 6.5). After addition of SDS buffer containing 10%β-mercaptoethanol (0.125 M Tris-HCl, 4% sodium dodecylsulfate, 20%glycerol, 0.002% Bromophenol Blue, pH 6.8), the mixture was heated for 5minutes at 100° C. and subjected to SDS-PAGE (12.5% polyacrylamide gel,Daiichi Pure Chemical Co.). The gel was fixed and dried according to aconventional method. After isotope signals were enhanced using Amplify™(Amersham Co.), the dried gel was subjected to autoradiography at −80°C. using Bio Max MR film (Kodak Co.). The results are shown in FIG. 18,which shows that the molecular weight of the protein encoded by the cDNAof the present invention is about 40,000.

Example 22 Binding of the Protein Encoded by cDNA of the PresentInvention and OCIF

PhOBM, which was purified in the same manner as in the Example 21(2),was transfected into COS-7 cells in each well of a 24-well plate usingLipofectamine. After cultured for 2 to 3 days, the cells were washedwith serum-free DMEM. Two hundred p1 of a culture medium for a bindingtest medium (serum-free DMEM to which 0.2% bovine serum albumin, 20 mMHepes buffer solution, 0.1 mg/ml heparin, and 0.2% NaN₃ were added)containing 20 ng/ml of ¹²⁵I-labeled OCIF was added to the wells. To theother wells, 200 of culture medium for the binding test mediumcontaining 8 μg/ml of unlabeled OCIF in addition to 20 ng/ml of¹²⁵I-labeled OCIF, was added. After incubation for one hour at 37° C. ina CO₂ incubator (5% CO₂), the cells were washed twice with 500 μl of aphosphate buffered saline solution containing 0.1 mg/ml of heparin.Then, 500 μl of 0.1 N NaOH solution was added to each well and the platewas allowed to stand for 10 minutes at room temperature to dissolve thecells. The radioactivity of ¹²⁵I in the wells was measured by a gammacounter. As a result, as shown in FIG. 19, it was confirmed that the¹²⁵I-labeled OCIF binds only to the cells transfected with phOBM.Moreover, the binding was significantly inhibited by adding 400-foldexcess unlabelled OCIF (8 μg/ml). Based on the results described above,the protein, human OBM encoded by the cDNA in the phOBM was confirmed tospecifically bind to OCIF on the surface of COS-7 cells.

Example 23 Crosslinking of ¹²⁵I-Labeled OCIF and the Protein Encoded bythe cDNA of the Present Invention

Crosslinking of ¹²⁵I-labeled monomer type OCIF and the protein encodedby the cDNA of the present invention was carried out to furtherinvestigate the characteristics of the protein encoded by the cDNA ofthe present invention. After constructing expression vector phOBM andtransfecting into COS-7 cells according to the method used in theExamples 21(1) and 21(2), 200 μl of binding test medium containing¹²⁵I-labeled OCIF (25 ng/ml) described above was added. The binding testmedium to which unlabeled OCIF was added at a 400-fold concentration inaddition to ¹²⁵I-labeled OCIF was used for the other wells. Aftercultured for one hour at 37° C. in a CO₂ incubator (5% CO₂), the cellswere washed twice with 500 μl of phosphate buffered saline containing0.1 mg/ml heparin. Five hundred μl of phosphate buffered saline in which100 μg/ml of a crosslinking agent (DSS: disuccinimidyl suberate,manufactured by Pierce Co.) dissolved was added to the cells, followedby incubation for 10 minutes at 0° C. The cells in these wells werewashed twice with 1 ml of ice-cold phosphate buffered saline. After anaddition of 100 μl of 20 mM Hepes buffer solution containing 1% TritonX-100 (Wako Pure Chemicals Co., Ltd.), 2 mM PMSF (Phenylmethylsulfonylfluoride, Sigma Co.), 10 μM Pepstatin (Wako Pure Chemicals Co., Ltd.),10 μM leupeptin (Wako Pure Chemicals Co., Ltd.), 10 μM antipain (WakoPure Chemicals Co., Ltd.) and 2 mM EDTA (Wako Pure Chemicals Co., Ltd.),the wells were allowed to stand for 30 minutes at room temperature todissolve the cells. These samples (15 aliquots) were treated with SDSunder reducing conditions according to a conventional method andsubjected to SDS-electrophoresis using 4-20% polyacrylamide gradient gel(Daiichi Pure Chemical Co., Ltd.). After electrophoresis, the gel wasdried and subjected to autoradiography for 24 hours at −80° C. usingBioMax MS film (Kodak Co.) and BioMax MS sensitization screen (KodakCo.). The film subjected to autoradiography was developed according to aconventional method. As a result, a band of a molecular weight in therange of 90,000-110,000, shown in FIG. 20, was detected by crosslinkingof ¹²⁵I-labeled monomer type OCIF and the protein encoded by the cDNA ofthe present invention.

Example 24 Expression of Secreted-Form Human OBM (1) Construction ofSecreted-Form Human OBM Expression Plasmid

A PCR was carried out using human OBM SF (SEQ ID NO:13) and mouse OBM #8(SEQ ID NO:6) as primers and pUC19hOBM as a template. After purificationby electrophoresis on an agarose gel, the product was digested withrestriction enzymes SPlI and HindIII, and further purified byelectrophoresis on an agarose gel to obtain a purified fragment (0.27kb). Human OBM cDNA was partially digested with restriction enzyme DraIand DNA fragments digested with DraI at one site were purified byelectrophoresis on an agarose gel. The purified fragment was furtherdigested with restriction enzyme HindIII. The 0.53 kb DraI/HindIIIfragment was purified by electrophoresis on an agarose gel. The purifiedfragment was ligated with the 0.27 kb SplI/HindIII fragment derived fromthe PCR described above using ligation kit ver. 2 (Takara Shuzo Co.)together with HindIII/EcoRI fragment (5.2 kb) of pSec TagA (InvitrogenCo.). Escherichia coli DH5α was transformed using the reaction product.Plasmids were purified by alkali SDS method from the resultingampicillin resistant transformants and digested with restriction enzymesto select a plasmid containing 0.27 kb and 0.53 kb-fragments as insertsin pSec TagA. This plasmid was confirmed to have a sequence encoding thesecreted human OBM by sequencing using Tag dyedeoxyterminator cyclesequencing FS kit (Perkin Elmer Co.). The plasmid was digested withrestriction enzymes Nhel and Xhol to prepare a fragment (0.8 kb)corresponding to the secreted human OBM cDNA by electrophoresis on anagarose gel. This fragment was inserted into the NheI and XhoI fragment(10.4 kb) of an expression vector pCEP4 (Invitrogen Co.) using ligationkit and Escherichia coli DH5α was transformed using the reactionproduct. Plasmids were purified by alkali-SDS method from the resultingampicillin resistant transformants and digested with restriction enzymesto select an Escherichia coli having the expression plasmid forsecreted-form human OBM (pCEPshOBM). The Escherichia coli containing thepCEPshOBM was cultured and pCEPshOBM was purified using a Qiafilterplasmid midi kit (Qiagen Co.).

(2) Expression of Secreted-Form OBM

293-EBNA cells were suspended in IMDM containing 10% FCS (IMDM-10% FCS),added into a 24-well plate coated with collagen (manufactured bySumitomo Bakelite Co., Ltd.) in a cell density of 2×10⁵/2 ml/well andcultured overnight. The cells were transfected with 1 μg of pCEPshOBM orpCEP4 using 4 μl of Lipofectamine (Gibco Co.). After cultured for twodays in 0.5 ml of a serum-free IMDM or IMDM-10% FCS, the culturesupernatants were collected. Expression of the secreted human OBM in theculture supernatant was detected as follows. Sodium bicarbonate wasadded to the culture supernatants to a final concentration of 0.1 M andthe mixtures were added to a 96-well plate. The plate was allowed tostand overnight at 4° C., thereby immobilizing human OBM in the culturesupernatants on the 96-well plate. The plate was blocked using Block Ace(Snow Brand Milk Products Co., Ltd.) solution four-fold diluted with PBS(B—PBS) and allowed to stand for two hours at room temperature. Afteradding 3-100 ng/ml of OCIF which was diluted with B—PBS to each well,the plate was allowed to stand for two hours at 37° C., followed by washwith PBS containing 0.05% Polysolvate 20(P—PBS). Then, 100 μl of aperoxidase-labeled rabbit anti-OCIF polyclonal antibody described in WO96/26217 which was diluted with B—PBS was added to each well. Afterallowing to stand for two hours at 37° C., the wells were washed sixtimes with P—PBS. Then, TMB solution (TMB Soluble Reagent, HighSensitivity, Scytek Co.) was added in the amount of 100 μl per well andthe mixture was allowed to stand at room temperature for about 10minutes. The reaction was terminated by the addition of 100 μl oftermination solution (Stopping Reagent, Scytek Co.) to each well.Absorbance at 450 nm for each well was measured by a microplate reader.The results are shown in FIG. 21, which indicates that the absorbance at450 nm increased according to the concentration of the added OCIF in theplate in which the conditioned medium of the cells transfected withpCEPshOBM was immobilized. On the other hand, no increase in absorbancewas seen in the wells in which the conditioned medium of the cellstransfected with vector pCEP4 was immobilized. FIG. 22 shows the resultsof an experiment wherein the proportion of the conditioned medium usedfor immobilization was changed within a range of 5-90% in the presenceof a constant concentration of OCIF (50 ng/ml). The absorbance at 450 nmincreased according to the increase in the proportion of the conditionedmedium in the plate wherein the conditioned medium of the cellstransfected with pCEPshOBM was immobilized, whereas no such increase inabsorbance was seen in the plate wherein the conditioned medium of thecells transfected with vector pCEP4 was immobilized. From these results,it was confirmed that secreted-form human OBM is produced in theconditioned medium of the cells transfected with pCEPshOBM.

Example 25 Expression of Thioredoxin-Human OBM Fusion Protein (Trx-hOBM)(1) Construction of a Thioredoxin-Human OBM Fusion Protein (Trx-hOBM)Expression Vector

Ten μl of 10′ExTaq buffer (Takara Shuzo Co.), 8 μl of 10 mM dNTP (TakaraShuzo Co.), 77.5 μl of sterilized distilled water, 2 μl of an aqueoussolution of pUC19hOBM (10 ng/μl), 1 μl of primer, mouse OBM #3 (100pmol/μl, SEQ ID NO:9), 1 of primer, hOBM SalR2 (100 pmol/μl, SEQ IDNO:10), and 0.5 μl of ExTaq (5 u/μl) (Takara Shuzo Co.) were mixed andreacted (PCR) in a micro centrifuge tube. After the reaction at 95° C.for 5 minutes, at 50° C. for one second, at 55° C. for one minute, at74° C. for one second, and at 72° C. for 5 minutes, a cycle reactionconsisting of a reaction at 96° C. for one minute, at 50° C. for onesecond, at 55° C. for one minute, at 74° C. for one second, and at 72°C. for 3 minutes, was repeated 25 times. From the total reaction mixtureDNA fragment (750 bp) was purified. The whole amount of purified DNAfragment was digested with restriction enzymes SaII (Takara Shuzo Co.)and BspHI (New England Bilabs Co.), and subjected to electrophoresis ona 1% agarose gel to obtain purified DNA fragment (Fragment 1, about 320bp). The fragment was dissolved in 20 μl of sterilized distilled water.In the same manner, DNA fragment (Fragment 2, about 450 bp) obtained bydigesting 4 μg of pUC19hOBM with restriction enzymes BamHI, and BspHI(Takara Shuzo Co.) and DNA fragment (Fragment 3, about 3.6 kb), obtainedby digesting 2 μg of pTrXFus (Invitrogen Co.) with restriction enzymesBamHI, and SaII (Takara Shuzo Co.) were respectively purified anddissolved in 20 μl of sterilized distilled water. The QIAEXII gelextraction kit was used for purification of the DNA fragments. Fragments1-3 were ligated by incubating at 16° C. for 2.5 hours using DNAligation kit ver.2 (Takara Shuzo Co.). Using the ligation reaction,Escherichia coli GI724 (Invitrogen Co.) was transformed according to themethod described in the Instruction Manual of ThioFusion ExpressionSystem (Invitrogen Co.). A microorganism strain with plasmid in whichthe hOBM cDNA fragment is fused in frame to thioredoxin gene wasselected from the resulting ampicillin resistant transformants byanalysis of DNA restriction map obtained by digestion with restrictionenzyme and by determination of DNA sequence. The microorganism strainthus obtained was named GI724/pTrxhOBM.

(2) Expression of Trx-hOBM in Escherichia coli

GI724/pTrxhOBM and GI724 containing pTrxFus (GI724/pTrxFus) wererespectively cultured six hours with shaking at 30° C. in 2 ml ofRMG-Amp medium (0.6% Na₂HPO₄, 0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 2%casamino acid, 1% glycerol, 1 mM MgCl₂, 100 μg/ml ampicillin, pH 7.4).The broth (0.5 ml) was added to 50 ml of Induction medium (0.6% Na₂HPO₄,0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 0.2% casamino acid, 0.5% glucose, 1mM MgCl₂, 100 μg/ml ampicillin, pH 7.4) and cultured with shaking at 30°C. When OD₆₀₀ nm reached about 0.5, L-tryptophan was added to a finalconcentration of 0.1 mg/ml, followed by culturing with shaking at 30° C.for an additional 6 hours. The culture broth was centrifuged at 3000×gto collect the cells, which were then suspended in 12.5 ml of PBS. Thesuspension was subjected to an ultrasonic generator (Ultrasonics Co.) todisrupt the cells. The disrupt cells were centrifuged at 7000×g for 30minutes to obtain a supernatant liquid as a soluble protein fraction.Ten μl of this soluble protein fraction was subjected to SDSpolyacrylamide (10%) electrophoresis under reducing conditions. As aresult, as shown in FIG. 23, a band with a molecular weight of 40,000which was not detected in the soluble protein fraction of GI724/pTrxFuswas found in the soluble protein fraction of GI724/pTrxhOBM.Accordingly, it was confirmed that a fusion protein (Trx-hOBM) ofthioredoxin and human OBM was expressed in Escherichia coli.

(3) Binding Capability of Trx-hOBM to OCIF

Binding of the expressed Trx-hOBM to OCIF was confirmed according to thefollowing experiment. Anti-thioredoxin antibody (Invitrogen Co.) whichwas diluted 5000-fold with 10 mM sodium hydrogen carbonate solution wasadded to a 96-well immunoplate (Nunc Co.) in the amount of 100 μl perwell. After being allowed to stand overnight at 4° C., the liquid in thewells was discarded. Two hundred μl of a solution prepared by dilutingBlock Ace (Snow Brand Milk Products Co., Ltd.) two-fold with PBS(BA-PBS) was added to each well. After being allowed to stand for onehour at room temperature, the wells were washed three times with P—PBS.The soluble protein fractions originating from the above-describedGI724/pTrxhOBM or GI724/pTrxFus, each diluted with BA-PBS in variousconcentrations were added to each well in the amount of 100 μl. Afterbeing allowed to stand for two hours at room temperature, each well waswashed three times with P—PBS and charged with 100 μl of OCIF (100ng/ml) which was diluted with BA-PBS. After being allowed to stand fortwo hours at room temperature, each well was washed three times withP—PBS and charged with 100 μl of peroxidase-labeled anti-OCIF antibody(described in WO 96/26217) which was diluted 2,000-fold with BA-PBS.After being allowed to stand for two hours at room temperature, eachwell was washed six times with P—PBS and charged with 100 μl of TMBsolution. After being allowed to stand for about 10 minutes at roomtemperature, each well was charged with 100 μl of termination solution(Stopping Reagent). Absorbance of each well at 450 nm was measured by amicroplate reader. The results are shown in FIG. 24. There was nodifference in the absorbance between the sample with the soluble proteinfraction originating from GI724/pTrxFus added thereto and the samplewithout the addition of this soluble protein fraction. On the otherhand, the absorbance increased in the samples to which the solubleprotein fraction originating from GI724/pTrxhOBM was added in proportionto the concentration of the soluble protein fraction. The results of theother experiment wherein the dilution rate of the soluble proteinfraction was maintained constant (1%) while adding OCIF diluted withBA-PBS in different concentrations (0-100 ng/ml) are shown in FIG. 25.It can be seen that the absorbance remained low at any concentrations ofOCIF in samples using a soluble protein fraction originating fromGI724/pTrxFus, whereas the absorbance increased in proportion to theOCIF concentration in the samples to which the soluble protein fractionoriginating from GI724/pTrxhOBM was added. Based on these results, itwas confirmed that Trx-hOBM which is produced from GI724/pTrxhOBM has acapability of binding to OCIF.

(4) Large-Scale Cultivation of Escherichia coli Which Produces Trx-hOBM

GI724/pTrxhOBM cells were spread on RMG-Amp agar (0.6% Na₂HPO₄, 0.3%KH₂PO₄, 0.05% NaCl, 0.1% NH₄Cl, 2% casamino acid, 1.5% agar, pH 7.4)using a platinum transfer 100 p. The cells were cultured overnight at30° C. The cultured cells were suspended in 10 ml of Induction medium.The suspension was added (5 ml for each) to two 21 Erlenmeyer flaskscontaining 500 ml of Induction medium and cultured at 30° C. withshaking. When the OD₆₀₀ nm reached about 0.5, L-tryptophan was added toa final concentration of 0.1 mg/ml. Culturing with shaking was continuedfor six hours at 30° C. The culture broth was centrifuged for 20 minutesat 3000×g to collect the cells, which were suspended in 160 ml of PBS.The suspension was subjected to an ultrasonic generator (UltrasonicsCo.) to disrupt the cells. The supernatant liquid was centrifuged for 30minutes at 7000×g to obtain a soluble protein fraction.

(5) Preparation of OCIF-Immobilized Affinity Column

Two g of TSKgel AF-Tolesyl Toyopal 650 (Tosoh Corp.) and 40 ml of 1.0 Mpotassium phosphate buffer (pH 7.5) containing 35.0 mg of recombinantOCIF, which was prepared according to the method described in WO96/26217, were mixed. The mixture was gently shaken overnight at 4° C.to effect a coupling reaction. The reaction mixture was centrifuged toremove the supernatant. To inactivate excess active residues, 40 ml of0.1 M Tris-HCl buffer (pH 7.5) was added to the precipitated carrier andthe mixture was gently shaken at room temperature for one hour. Thecarrier in a column was washed with 0.1 M glycine-HCl buffer (pH 3.3)containing 0.01% Polysorbate 80 and 0.2 M NaCl and 0.1 M sodium citratebuffer (pH 2.0) containing 0.01% Polysorbate 80 and 0.2 M NaCl. Thecarrier in the column was equilibrated by charging twice with 10 mMsodium phosphate buffer (pH 7.4) containing 0.01% Polysorbate 80.

(6) Purification of Trx-hOBM Using OCIF-Immobilized Affinity Column

Unless otherwise indicated, purification of Trx-hOBM was carried out at4° C. The above-mentioned OCIF-immobilized affinity carrier (10 ml) andthe soluble protein fraction (120 ml) prepared in Example 25(4) weremixed. The mixture was gently shaken overnight at 4° C. in four 50 mlcentrifuge tubes using a rotor. An Econo-column™ (internal diameter: 1.5cm, length: 15 cm, manufactured by BioRad Co.) was filled with thecarrier in the mixture. The column was charged with 300 ml of PBScontaining 0.01% Polysorbate 80, 100 ml of 10 mM sodium phosphate buffer(pH 7.0) containing 0.01% Polysorbate 80 and 2.0 M NaCl, and 100 ml of0.1 M glycine-HCl buffer (pH 3.3) containing 0.01% Polysorbate 80 and0.2 M NaCl, in that order. Next, proteins adsorbed in the column wereeluted with 0.1 M sodium citrate buffer (pH 2.0) containing 0.01%Polysorbate 80 and 0.2 M NaCl. The eluate was collected in 5 mlportions. Each fraction thus collected was immediately neutralized withaddition of 10% volume of 2 M Tris buffer solution (pH 8.0). Presence orabsence of Trx-hOBM in the eluted fractions was determined according tothe method previously described in Example 25(3) (the binding capabilityto OCIF). The fractions containing Trx-hOBM were collected and purifiedfurther.

(7) Purification of Trx-hOBM by Gel Filtration

About 25 ml of Trx-hOBM fractions obtained in Example 25(6) wasconcentrated to about 0.5 ml by centrifuging using Centriplus 10 andCentricon 10 (Amicon Co.). This sample was applied to a Superose 12 HR10/30 column (1.0×30 cm, Pharmacia Co.) previously equilibrated with PBScontaining 0.01% Polysorbate 80. For the separation, PBS containing0.01% Polysorbate 80 was used as a mobile phase at a flow rate of 0.25ml/min. The eluate from the column was collected in 0.25 ml portions.The Trx-hOBM in the thus-collected fractions was detected by the samemethod as previously described in the Example 25(3) and SDS-PAGE.Fractions containing purified Trx-hOBM were collected and the proteinconcentration of Trx-hOBM was determined. The measurement of the proteinconcentration was carried out using bovine serum albumin as a standardsubstance using DC-Protein assay kit (BioRad Co.).

Example 26 Osteoclast Formation-Inducing Activity of hOBM

phOBM and pcDL-SRα296 were respectively transfected into COS-7 cellsusing Lipofectamine (Gibco Co.). The cells were cultured for one day inDMEM containing 10% FCS, trypsinized, plated on cover slips (15 mm roundshape, manufactured by Matsunami Co.) in 24-well plates at 5×10⁴ cellsper well, and cultured for 2 days. The culture plate was washed oncewith PBS. The cells were fixed with PBS containing 1% paraformaldehydeat room temperature for 8 minutes. The plate on which the fixed cellswere attached was washed 6 times with PBS, then 700 μl of mouse spleencells suspended at 1×10⁶/ml in α-MEM containing 10⁻⁸ M active-formvitamin D₃, 10⁻⁷ M dexamethasone, and 10% fetal bovine serum were addedto each well. Millicell PCF (Millipore Co.) was set in each well and asuspension of ST2 cells in the above-mentioned culture medium (4×10⁴/ml)were added, 700 μl per well, into the Millicell PCF followed byincubation at 37° C. for 6 days. After the culture, the Millicell PCFwas removed, the plate was washed once with PBS, and the cells werefixed with acetone-ethanol solution (50:50) for one minute. Then, thecells exhibiting tartaric acid-resistant acid phosphatase activity(TRAP), which is a specific marker for osteoclast, were selectivelystained using LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.). As a result ofmicroscopic observation, TRAP-positive cells were not detected in thewells in which COS-7 cells transfected with pcDL-SR α296 were fixed. Incontrast, 65±18 (average±standard deviation, n=3) TRAP positive cellswere observed in the wells in which COS-7 cells transfected with phOBMwere fixed. Moreover, expression of calcitonin receptor was confirmed bythe fact that ¹²⁵I-labeled salmon calcitonin (Amersham Co.) specificallybound to these TRAP positive cells. Based on these findings, it has beenproven that human OBM, which is the protein encoded by cDNA of thepresent invention, has osteoclast formation-inducing activity.

Example 27 Osteoclast Formation-Inducing Activity of Trx-hOBM andSecreted-Form Human OBM

Mouse spleen cells were suspended inα.-MEM containing 10⁻⁸ M active-formvitamin D₃, 10⁻⁷ dexamethasone, and 10% fetal bovine serum at aconcentration of 2×10⁶/ml. The suspension was added to a 24 well platein the amount of 350 μl per well. Each well was then charged with 350 μlof a solution prepared by diluting purified Trx-hOBM with theabove-mentioned culture medium (40 ng/ml), 350 μl of solution preparedby 10-fold diluting a conditioned medium which was produced by culturing293-EBNA cells, onto which pCEPshOBM or pCEP4 were transfected, in aculture medium IMDM-10% FCS, with above-mentioned culture medium, or 350μl only of the above-mentioned culture medium. The Millicell PCF(Mollipore Co.) was placed on each well, to which 600 μl of ST2 cellswhich were suspended in the above-mentioned culture medium (4×10⁴/ml)were added. After cultured for six days, the Millicell PCF was removed.The plate was washed once with PBS and the cells were fixed byacetone-ethanol solution (50:50) for one minute. Then, the cellsexhibiting the activity of tartaric acid resistant acidic phosphatase(TRAP activity) were selectively stained using LEUKOCYTE ACIDPHOSPHATASE kit (Sigma Co.). The results of microscopic observationrevealed that no cells exhibiting the TRAP activity were detected in thewells to which no Trx-hOBM was added, whereas 115±19 (average±standarddeviation, n=3) TRAP-positive cells were observed in the wells to whichTrx-hOBM was added. Similarly, while no cells exhibiting TRAP activitywere detected in the wells to which conditioned medium of 293-EBNA cellstransfected with pCEP4 had been added, 125±23 (average±standarddeviation, n=3) TRAP positive cells were observed in the wells to whichconditioned medium of 293-EBNA cells transfected with pCEPshOBM had beenadded. Moreover, expression of calcitonin receptor was confirmed by thefact that ¹²⁵I-labeled salmon calcitonin (Amersham Co.) specificallybinds to these TRAP positive cells. These results have proven thatTrx-hOBM and secreted-form hOBM exhibit osteoclast formation-inducingactivity.

Example 28 Preparation of Polyclonal Antibody

Mouse sOBM or human sOBM used as an immunogen was prepared according tothe method described above. Especially, mouse sOBM cDNA (cDNA (SEQ IDNO:18) encoding mouse sOBM (SEQ ID NO:16) which does not have themembrane binding region of the mouse OBM due to absence of the aminoacids from the N-terminal down to the 72nd amino acid) or human sOBMcDNA (cDNA (SEQ ID NO:19) encoding human sOBM (SEQ ID NO:17) which doesnot have the membrane binding region of human OBM due to absence of theamino acids from the N-terminal down to the 71st amino acid) was ligatedwith a Hind III/EcoRV fragment (5.2 kb) of the expression vector pSecTagA (Invitrogen Co.) including the nucleotide sequence encoding asignal peptide of K-chain of immunoglobulin, together with anEcoRI/PmaCl fragment (0.32 kb) of OBM cDNA, using a ligation kit ver. 2(Takara Shuzo Co.). Escherichia coli DH5α was transformed with thereaction product. The plasmids obtained from the resulting ampicillinresistant strains were purified by the alkali SDS and digested with anrestriction enzyme to select a plasmid with 0.6 Kb and 0.32 kb fragmentsinserted into pSec TagA. The sequence of this plasmid was identifiedusing the Dyedeoxyterminator Cycle Sequencing FS kit (product of PerkinElmer Co.). As a result, it was confirmed that this plasmid has asequence encoding mouse or human sOBM. After plasmid was digested withrestriction enzymes NheI/XhoI, a fragment (1.0 kb) corresponding tosecretion form OBM cDNA was recovered by agarose gel electrophoresis.This fragment was inserted into an NheI/XhoI fragment (10.4 kb) of theexpression vector pCEP4 (Invitrogen Co.) using a ligation kit.Escherichia coli DH5α was transformed using the reaction product.Plasmids were purified by the alkali SDS from the resulting ampicillinresistant strains. Analyzing these plasmids by digesting with arestriction enzyme, Escherichia coli possessing a secretion type OBMexpression plasmid (pCEP sOBM) having the objective structure wasselected. The Escherichia coli strain having the pCEP sOBM was culturedand pCEP sOBM was purified using a Qiafilter plasmid midi kit (QiagenCo.). Next, 293-EBNA cells were suspended in IMDM (IMDM-10% FCS)containing 10% FCS and plated onto a 24-well plate coated with collagen(product of Sumitomo Bakelite Co., Ltd.) at a cell density of 2×10⁵cells/2 ml/well. After culturing overnight, the cells were transformedwith 1 μg of pCEP sOBM or pCEP4 using 4 μl of Lipofectamine (Gibco Co.)and further cultured for two days in 0.5 ml of serum-free IMDM orIMDM-10% FCS. The culture supernatant was recovered. A cell line withhigh productivity of recombinant mouse soluble OBM (msOBM) or humansoluble OBM (hsOBM) was screened as follows. Sodium bicarbonate wasadded to the culture supernatant which is assumed to contain msOBM orhsOBM to a final concentration of 0.1 M. One hundred p1 of the culturesupernatant was added to each well in 96-well immunoplates (Nunc Co.)and allowed to stand overnight at 4° C., thereby msOBM or hsOBM in theculture supernatant was immobilized on each well. To each well, 200 μlof Block Ace (Snow Brand Milk Products Co., Ltd.) solution dilutedfour-fold with PBS (B—PBS) was added and the plates were allowed tostand for two hours at room temperature. After washing each well in theplates three times with PBS (P—PBS) containing 0.1% Polysorbate 20, 100μl of each recombinant OCIF (rOCIF) solution (3-100 ng/ml) dilutedserially with P—PBS was added to each well in the plates. The plateswere allowed to stand for two hours at 37° C. After washing the platesthree times with PBS, 100 μl of a peroxidase-labeled anti-OCIFpolyclonal antibody (WO 96/26217) diluted with B—PBS was added to eachwell. After allowing to stand for two hours at 37° C., the wells werewashed six times with P—PBS. Then, 100 μl of TMB solution (TMB SolubleReagent, High Sensitivity, ScyTek Co.) was added to each well in theplates and the plates were allowed to stand at room temperature forabout 10 minutes, subsequently the reaction was terminated by adding 100μl of a stopping solution (Stopping Reagent, ScyTek Co.) to each well.Absorbance at 450 nm of each well was measured using a microplatereader. It was confirmed that the absorbance increased remarkably inproportion to concentration of the added OCIF in the plates in whichmsOBM or hsOBM in the culture supernatant of the cell line producingmsOBM or hsOBM was immobilized therein.

The cell line that exhibited a high rate of increase in absorbance wasselected as a strain with high productivity. Thus-related 293-EBNA cellswith high productivity of msOBM or hsOBM were cultured on a large scalein an IMDM medium containing 5% FCS, using 25 T-flasks (T-225). Afterthe cell reached to confluent, a fresh culture medium was added to eachT-225 flask in the amount of 100 ml per flask and cells were culturedfor 3-4 days, to collect the culture supernatant. These procedures wererepeated four times to obtain 10 L of the culture supernatant containingmsOBM or hsOBM. Purified msOBM (10 mg) or hsOBM (12 mg), which showshomogeneous band (molecular weight: 32 kDa) on SDS-polyacrylamide gelelectrophoresis, were obtained from the culture supernatant by means ofaffinity chromatography on an OCIF-immobilized column and gel filtrationchromatography according to the method described in examples 25(6) and25(7). Each thus-obtained purified preparation was used as an antigenfor immunization. Each protein antigen obtained was dissolved inphosphate buffered saline (PBS) to a concentration of 200 μg/ml andemulsified with an equivalent volume of Freund's complete adjuvant. Oneml of the emulsion was subcutaneously immunized to each of threeJapanese white rabbits about once every week. A booster injection wasgiven when the antibody titer reached a peak. Whole blood was collected10 days thereafter. The serum was diluted two-fold with a binding bufferfor protein A sepharose chromatography (BioRad Co.) and applied to aprotein A column equilibrated with the same buffer. After washing thecolumn extensively with the same buffer, the anti-sOBM antibody adsorbedto the column was eluted with an elution buffer (BioRad Co.) or 0.1 Mglycine-HCl buffer, pH 3.0. To neutralize the eluate immediately, theeluate was fractionated using test tubes containing a small amount of1.0 M Tris-HCl (pH 8.0). The eluate was dialyzed against PBS overnightat 4° C. The antibody content in the antibody solution was measured bythe Lowry method using bovine IgG as a standard protein. Thus, about 10mg of purified immunoglobulin (IgG) containing the polyclonal antibodyof the present invention per 1 ml of rabbit antiserum was obtained.

Example 29 Measurement of OBM and sOBM by ELISA Using PolyclonalAntibody

A sandwich ELISA was constructed using the rabbit anti-human sOBMpolyclonal antibody obtained in Example 28 as the solid phase antibodyand enzyme-labeled antibody. Peroxidase (POD)-labeled antibody wasprepared according to the method of Ishikawa (Ishikawa et al., J.Immunoassay, Vol. 4, 209-327, 1983).

The anti-human sOBM polyclonal antibody obtained in the Example 28 wasdissolved in a 0.1 M NaHCO₃ to a concentration of 2 μg/ml. One hundredp1 of the resulting solution was added to each well in 96-wellimmunoplates (Nunc Co.), which was then allowed to stand at roomtemperature overnight. Next, 200 μl of 50% Block Ace (Snow Brand MilkCo., Ltd.) was added to each well and the plates were allowed to standfor one hour at room temperature. The wells were washed three times withPBS containing 0.1% Polysorbate 20 (washing buffer).

Human OBM was expressed according to the method of Example 26 andpurified according to the method of Example 2. The purified human OBMand the purified human sOBM prepared in example 28 were serially dilutedwith the first reaction buffer (0.2 M Tris-HCl buffer, pH 7.2,containing 40% Block Ace and 0.1% Polysorbate 20), respectively, and 100ml of the diluted solution was added to each well in the plates. Theplates were allowed to stand at room temperature for two hours, andwashed three times with the above-mentioned washing buffer.Subsequently, 100 μl of POD-labeled anti-human sOBM polyclonal antibodydiluted 1000-fold with the second reaction buffer (0.1 M Tris-HClbuffer, pH 7.2, containing 25% Block Ace and 0.1% Polysorbate 20) wasadded to each well in the plates. After the plates were allowed to standat room temperature for two hours, each well was washed three times withthe washing buffer. Next, 100 μl of enzyme substrate solution (TMB,ScyTek Co.) was added to each well in the plates, and the plates wereallowed to stand for 10 minutes, followed by the addition of 100 μl of areaction termination solution (Stopping reagent, ScyTek Co.) to stop theenzyme reaction. The absorbance at 450 m of each well was measured usinga microplate reader. The results are shown in FIG. 26. The sandwichELISA using a rabbit anti-human sOBM polyclonal antibody recognizedalmost equally human sOBM (molecular weight, about 32 kDa) and human OBM(molecular weight, about 40 kDa), with a measurement sensitivity ofabout 12.5×10⁻³ μmol/ml (human OBM: about 500 pg/ml, human sOBM: about400 μg/ml). The measurement of mouse sOBM and mouse OBM by ELISA usingthe rabbit anti-mouse sOBM polyclonal antibody obtained in the Example28 was able to be carried out in the same manner. It was confirmed thatan extremely small amount of mouse sOBM or mouse OBM can be measuredwith almost the same sensitivity as described above.

As mentioned above, the anti-human sOBM polyclonal antibody of thepresent invention prepared in the Example 28 can equally recognize boththe human sOBM and human OBM antigens. Therefore, the antibody was namedanti-human OBM/sOBM polyclonal antibody. Similarly, the anti-mouse sOBMpolyclonal antibody prepared in the Example 28 can equally recognizeboth the mouse sOBM and mouse OBM antigens. This antibody was thereforenamed anti-mouse OBM/sOBM polyclonal antibody.

Example 30 Preparation of Monoclonal Antibody

The purified human sOBM prepared in the Example 28 was used as theantigen for immunization. The purified human sOBM was dissolved inphysiological saline solution to a concentration of 10 μg/ml andemulsified by mixing with an equivalent volume of Freund's completeadjuvant. The emulsion was intraperitoneally administered to BALB/c miceat a dose of 200 μl three times, once a week, to immunize mice. Next,the equivalent volume of the Freund's complete adjuvant was added to aphysiological saline solution containing 5 μg/ml of human sOBM and themixture was sufficiently emulsified. This emulsion was injectedintraperitoneally to BALB/c mice at a dose of 200 μl, once a week forfour weeks for immunization. One week after the fourth immunization, 100μl of a physiological saline solution containing 10 μg/ml of human sOBMwas intravenously administered to the BALB/c mice as a booster. Afterthree days, the spleen was extracted and spleen cells were separated.The spleen cells were fused with mouse myeloma cells, P3×63-Ag8.653according to a conventional method (Koehler, G. and Milstein, C.,Nature, 256, 495 (1975)). The suspended fused cells were cultured for 10days in an HAT medium containing hypoxanthine, aminopterin, andthymidine. After the myeloma cells were dead and hybridomas appeared,the HAT medium was replaced with an aminopterin-free HAT medium, and thecell culture was continued.

Example 31 Selection of Hybridoma and Cloning

Appearance of hybridomas was recognized 10 days after cell fusion inExample 30. Monoclonal antibodies recognizing the human sOBM with highaffinity and hybridomas producing these antibodies were selectedaccording to the following procedure using the improved solid phaseELISA which is described below. In addition, to select the anti-OBMmonoclonal antibody which recognizes both human sOBM and mouse sOBM,mouse sOBM prepared in the Example 27 was used in addition to human sOBMas the antigen for the solid phase ELISA. The human sOBM and mouse sOBMwere respectively dissolved in a 0.1 M sodium bicarbonate solution at aconcentration of 5 μg/ml. Fifty ml of each antigen solution was added toeach well in 96-well immunoplates (Nunc Co.). The plates were allowed tostand at 4° C. overnight to immobilize the antigens. The antigensolution in each well was discarded. Each well was then filled with 200μl of 50% Block Ace (Snow Brand Milk Products Co., Ltd.) and allowed tostand at room temperature for one hour. After each well was washed withphosphate buffered saline solution (PBS—P) containing 0.1% Polysorbate20, 40 μl of calf serum (Hiclone Inc.) was added to each well.Subsequently, 10 μl of each hybridoma culture supernatant was added toeach well and each well was incubated at room temperature for two hoursin the presence of 80% calf serum. The object of the solid phase ELISAin the presence of 80% calf serum is to select a hybridoma which producean antibody which can detect a very small amount of human sOBM or mousesOBM even in a solution containing high concentration of protein and inthe presence of an immunoreaction interfering substance derived fromserum, i.e. a hybridoma which can produce an antibody with a highaffinity for human sOBM or mouse SOBM. After the reaction at roomtemperature for two hours, the plates were washed with PBS—P andsubsequently, 50 μl of peroxidase-labeled anti-mouse IgG (KPL Co.)diluted 5000-fold with physiological saline solution containing 25%Block Ace was added to each well. After the reaction at room temperaturefor two hours, the plate was washed three times with PBS—P. After theaddition of 50 μl of an enzyme substrate solution (TMB, ScyTek Co.) toeach well, the reaction was continued at room temperature for fiveminutes. The enzymatic reaction was stopped by the addition of 50 μl ofa termination solution (stopping reagent, ScyTek Co.). Hybridomas whichproduce antibodies recognizing human sOBM or mouse sOBM were selected bymeasuring absorbance at 450 nm of each well using a microplate reader(Immune Reader NJ2000™, Nippon InterMed. Co.). Hybridomas producingantibodies exhibiting particularly high absorbance (OD₄₅₀ nm) wereselected. Cloning of these hybridomas by a limiting dilution method wasrepeated 3 to 5 times to establish stable hybridomas. Hybridomasexhibiting particularly high antibody productivity were selected amongthe established antibody-producing hybridoma clones.

Example 32 Production and Purification of Monoclonal Antibody

The antibody-producing hybridomas obtained in the Example 31, i.e. highaffinity antibody-producing hybridoma which recognizes human sOBM andhybridoma which produces an antibody showing cross-reactivity to themouse sOBM were cultured, respectively. Each hybridoma was implantedintraperitoneally to BALB/c mice (1×10⁶ cells per mouse) to whichpristan (Aldorich Co.) was administered one week previously. After about2-3 weeks, accumulated ascites were collected. The monoclonal antibody,which recognizes human sOBM of the present invention or both the humansOBM and mouse sOBM in the ascites, was purified according to thepurification method of anti-OBM/sOBM polyclonal antibodies using aProtein A column described in the Example 28. The purified monoclonalantibody was thus obtained from the ascites by Protein A columnchromatography (Pharmacia Co.).

Example 33 Antigen Specificity of Monoclonal Antibody

The antigen specificity of a monoclonal antibody which specificallyrecognizes human sOBM and the monoclonal antibody exhibitingcross-reactivity to both the human sOBM and mouse sOBM was investigatedusing human sOBM, human intact OBM having a membrane binding region,mouse sOBM, and mouse intact OBM having a membrane binding region. Morethan thirty kinds of monoclonal antibody were obtained. The results onseveral representative antibodies are shown in Table 1. As a result, itwas found that most anti-human sOBM monoclonal antibodies whichspecifically recognize human sOBM also recognize the human intact OBMhaving a membrane binding region, but not the mouse OBM and the mouseintact OBM which has a membrane binding region. On the other hand, itwas found that only a few monoclonal antibodies recognizing both thehuman sOBM and mouse sOBM were obtained and that these antibodiesexhibit cross-reactivity to both the human OBM and mouse OBM. Theseresults show that there are common antigen-recognizing sites, namelycommon epitopes, in both the human OBM and mouse OBM. Based on the factthat the anti-human sOBM monoclonal antibody prepared using the humansOBM as an immune antigen also equally recognizes human OBM having amembrane binding region. Anti-human sOBM monoclonal antibody was namedthe anti-human OBM/sOBM monoclonal antibody.

TABLE 1 Antigen Antibody hsOBM hOBM MsOBM mOBM H-OBM 1 + + − − H-OBM2 + + − − H-OBM 3 + + − − H-OBM 4 + + − − H-OBM 5 + + − − H-OBM 6 + + −− H-OBM 7 + + − − H-OBM 8 + + − − H-OBM 9 + + + + H-OBM 10 + + − − H-OBM11 + + − − H-OBM 12 + + − − H-OBM 13 + + + + H-OBM 14 + + − − hsOBM:human soluble OBM, hOBM: human membrane bonding type OBM, msOBM: mousesoluble OBM, mOBM: mouse membrane bonding type OBM

Example 34 Determination of Class and Subclass of Monoclonal Antibody

The class and subclass of the monoclonal antibody of the presentinvention were determined by the immunoglobulin class and subclassanalysis kit (Amersham Co.) according to the protocol indicated. Theresults on representative monoclonal antibodies are shown in Table 2. Asshown in Table 2, the majority of anti-human OBM/sOBM monoclonalantibodies were IgG₁, the others were IgG_(2a) and IgG_(2b). Lightchains for all antibodies were κ chains.

TABLE 2 Antibody IgG₁ IgG_(2a) IgG_(2b) IgG₃ IgA κ H-OBM 8 − + − − − +H-OBM 9 + − − − − + H-OBM 10 + − − − − + H-OBM 11 + − − − − + H-OBM 12 −− + − − + H-OBM 13 + − − − − + H-OBM 14 + − − − − +

Example 35 Measurement of the Dissociation Constant (K_(d) value) ofMonoclonal Antibody

The dissociation constant of the monoclonal antibody was measuredaccording to a known method (Betrand Friguet et al.: Journal ofImmunological Methods, 77, 305-319, 1986). That is, the purifiedantibody obtained in the Example 32 was diluted with 0.4 M Tris-HClbuffer (a primary buffer, pH 7.4) containing 40% Block Ace and 0.1%Polysorbate 20 to give a concentration of 5 ng/ml. The solution wasmixed with an equivalent volume of a diluted solution of purified humansoluble OBM (hsOBM) obtained in Example 28 in the primary buffer with aconcentration range of 6.25 to 10 μg/ml. The mixture was allowed tostand for 15 hours at 4° C. to bind the hsOBM to the monoclonalantibody. After 15 hours, the antibody not bound to the hsOBM (10 μg/ml,100 μl/well) was measured using an immobilized solid phase ELISA tocalculate the dissociation constant of the monoclonal antibody to thehsOBM. In addition, affinity to msOBM of an antibody, which is amonoclonal antibody for the hsOBM and also exhibits the cross-reactivityto mouse soluble OBM (msOBM), was measured according to the same methodexcept for using msOBM instead of the hsOBM. Dissociation constant ofantibodies, which exhibit high affinity to each antigen and are usefulfor enzymatic immunoassay and binding assay, are shown in Table 3.

TABLE 3 Antibody Subclass Antigen Dissociation constant Kd(M) H-OBM 1IgG₁(κ) hsOBM 1 × 10⁻¹¹ < kd < 1 × 10⁻¹⁰ H-OBM 4 IgG₁(κ) hsOBM 1 × 10⁻¹¹< kd < 1 × 10⁻¹⁰ H-OBM 9 IgG₁(κ) hsOBM 1 × 10⁻⁹ < kd < 1 × 10⁻⁸ H-OBM 9IgG₁(κ) msOBM 1 × 10⁻⁸ < kd < 1 × 10⁻⁷

As a result, the dissociation constants (Kd) of H—OBM 1 and H—OBM 4which are the antibodies specific to human soluble OBM (hsOBM) were inthe order of 10⁻¹¹ M, indicating the high affinity to hsOBM. On theother hand, the Kd value of the antibody H—OBM 9 which recognizes boththe hsOBM and mouse soluble OBM (msOBM) was in the order of 10⁻⁸ M tomsOBM and in the order of 10⁻⁹ M to hsOBM. In addition, the dissociationconstant of the other antibody which recognizes both antigens in theTable 1, i.e. the dissociation constant of H—OBM 13 for each antigen,was the same as that of H—OBM 9, and these two antibodies belong to thesame subclass. These findings suggest the possibility that they are theidentical antibodies which recognize the same epitope of each antigen.

Example 36 Measuring Method of Human OBM and sOBM by Sandwich ELISAUsing Anti-Human OBM/sOBM Monoclonal Antibodies

A sandwich ELISA was constructed using the two high affinity monoclonalantibodies obtained in Example 35, H—OBM 1 and H—OBM 4, respectively asa solid phase antibody and an enzyme-labeled antibody. Labeling of theantibody was carried out using a maleimide activated-peroxidase kit(Piers Co.). The antibody, H—OBM 1, was dissolved in a 0.1 M sodiumbicarbonate solution to a concentration of 10 μg/ml, and 100 of thesolution was added to each well in 96-well immunoplates (Nunc company).After being allowed to stand overnight at 4° C. to immobilize theantibody, the solution was discarded and 300 μl of 50% Block Ace™solution was added to each well in the plates. Each well in the plateswas blocked by allowing to stand at room temperature for two hours.After blocking, the plates were washed with phosphate buffered salinecontaining 0.1% Polysorbate 20 (PBS—P). Human OBM (hOBM) and humansoluble OBM (hsOBM) were respectively diluted with 0.4 M Tris-HClbuffer, pH 7.4, containing 40% Block Ace™ (Snow Brand Milk Products Co.,Ltd.) and 0.1% Polysorbate 20 (Wako Pure Chemicals Co., Ltd.) (the firstreaction buffer) to prepare test samples with various concentrations.These test samples with different concentrations were added to each wellin the amount of 100 μl per well and reacted to the antibody, H—OBM 1immobilized on each well by incubating at room temperature for twohours. After two hours, the plates were washed with PBS—P. Next, 100 ofa solution of POD-labeled H—OBM 4 antibody in 0.2 M Tris-HCl buffer, pH7.4, containing 25% Block Ace™ and 0.1% Polysorbate 20 (the secondreaction buffer) was added to each well, followed by further incubatingat room temperature for two hours. The plates were then washed withPBS—P and 100 μl of an enzyme substrate solution (TMB, ScyTek Co.) wasadded to each well to start enzyme reaction. The enzyme reaction wasterminated by the addition of 100 μl of a reaction termination solution(stopping reagent, ScyTek Co.) to each well. The absorbance of each wellat 450 nm was measured using a microplate reader. The results are shownin FIG. 27.

As a result, it was confirmed that the sandwich ELISA constructed usingthe two anti-human OBM/sOBM monoclonal antibodies, H—OBM 1 and H—OBM 4with high affinity for human OBM/sOBM prepared in the Example 35,equally recognizes human OBM and human sOBM, and is able to measure avery small amount of human OBM or human sOBM at a quantitative limit ofabout 1.25×10⁻³ to 2.5×10⁻³ μmol/ml (about 50-100 μg/ml for human OBMwith a molecular weight of 40 kDa, about 40-80 μg/ml for human sOBM witha molecular weight of 32 kDa). The hybridomas which produce these twoanti-human OBM/sOBM monoclonal antibodies, H—OBM 1 and H—OBM 4 werenamed H—OBM1 and H—OBM4, respectively. The hybridoma producinganti-human OBM/sOBM monoclonal antibody (H—OBM 9) which recognizes mouseOBM and mouse sOBM and also has an osteoclast formation-inhibitoryactivity was named H—OBM9. These hybridomas were deposited with theNational Institute of Bioscience and Human Technology, the Agency ofIndustrial Science and Technology, on Nov. 5, 1993 with Deposition Nos.FERM BP-6264 (H—OBM 1), FERM BP-6265 (H—OBM 4), and FERM BP-6266 (H—OBM9).

Example 37 Measurement of Mouse OBM and Mouse sOBM Using Anti-HumanOBM/sOBM Monoclonal Antibody Which Recognizes Mouse OBM and Mouse sOBM

A sandwich ELISA was constructed using the anti-human OBM/sOBMmonoclonal antibody, H—OBM 9, which recognizes mouse OBM and mouse sOBMobtained as a solid phase antibody in the Examples 33 and 35, and theanti-mouse OBM/sOBM polyclonal antibody as an enzyme-labeled antibodyobtained in the example 28. The mouse OBM and mouse sOBM wererespectively diluted with the first reaction buffer to give variousconcentrations in the same manner as in the Example 35 and then measuredsOBM according to the method described in the Example 36. The resultsare shown in FIG. 28. As a result, it was found that mouse OBM and mousesOBM can be similarly measured using H—OBM 9 which is the anti-humanOBM/sOBM monoclonal antibody recognizing the mouse OBM and mouse sOBM ofthe present invention. As shown by the result of Example 35, thisanti-human OBM/sOBM monoclonal antibody H—OBM 9 has a high dissociationconstant relative to the mouse sOBM, namely it has a comparatively lowaffinity to mouse sOBM. The sensitivity in the measurement of mouse OBM(molecular weight, about 40 kDa) and mouse sOBM (molecular weight, about32 kDa) by this ELISA assay was about 25×10⁻³ μmol/ml (about 1 ng/ml formouse OBM and about 0.8 ng/ml for mouse sOBM).

Example 38 Osteoclastogenesis-Inhibitory Activity of Anti-OBM/sOBMAntibody

It is known that osteoclast-like cells (OCL) are induced by co-cultureof mouse spleen cells and ST2 cells (mouse bone marrow-derived stromalcells; Endocrinology, 125, 1805-1813 (1989)). Capability of theanti-OBM/sOBM antibody to inhibit the OCL formation when added to theco-culture system was studied. Because the mouse OBM is expressed inthis co-culture system, a rabbit anti-mouse OBM/sOBM polyclonal antibodywhich recognizes mouse OBM and an anti-human OBM/sOBM monoclonalantibody (H—OBM 9) which recognizes both human OBM and mouse OBMantigens were used as the antibodies in this example. Seven hundredmicroliters per well of each anti-OBM antibody diluted serially withα-MEM containing 10% FCS and 350 μl/well of male mouse splenocytes(2×10⁶/ml) suspended in the same medium described above were added toeach well in a 24-well plate (Nunc). Next, ST2 cells trypsinized andsuspended in the above-mentioned culture medium containing 4×10⁻⁸ MVitamin D₃ and 4×10⁻⁷ M Dexamethasone (8×10⁴ cells/ml) were added toeach well in the amount of 350 μl/well, followed by culturing for sixdays at 37° C. After the plates were washed once with PBS, cells in eachwell were fixed with a mixture of ethanol and acetone (50:50) for onehour at room temperature. The plates were dried in air, and 500 μl ofsubstrate solution was added to each well according to the protocol ofthe LEUKOCYTE ACID PHOSPHATASE kit (Sigma Co.), followed by incubatingfor 55 minutes at 37° C. Only the cells exhibiting the tartaricacid-resistant acid phosphatase activity (TRAP activity), which is aspecific marker for osteoclasts, were stained by this reaction. Theplates were washed once with distilled water and dried in air, and thenumber of TRAP-positive cells was counted. The results are shown inTable 4. As shown in Table 4, both the rabbit anti-mouse OBM/sOBMpolyclonal antibody and the anti-human OBM/sOBM monoclonal antibody,H—OBM 9, which recognizes mouse OBM inhibited OCL formation in adose-dependent manner. These antibodies were found to possessosteoclastogenesis-inhibitory activity likeosteoclastogenesis-inhibitory factor, OCIF/OPG, and thus are promisingas a therapeutic agent for treating bone metabolism abnormalitysymptoms.

TABLE 4 Number of TRAP-positive multinucleates Amount of Rabbit Mouseanti-human antibody anti-mouse OBM/sOBM OBM/sOBM monoclonal (μg/ml)polyclonal antibody antibody (H-OBM 9) 0 1155 ± 53  1050 ± 45  10 510 ±24 650 ± 25 100 10 ± 3 15 ± 4 (Average ± standard deviation, n = 3)

Example 39 Human Osteoclast Formation-Inducing Activity of Trx-OBM

Mononuclear cells were prepared from whole blood collected from the veinof a healthy adult by density gradient using Histopaque (Sigma Co.)according to the protocol attached thereto. The mononuclear cells weresuspended at a cell density of 1.3×10⁶/ml in α-MEM containing 10⁻⁷ MDexamethasone, 200 ng/ml macrophage colony stimulating factor (The GreenCross Corp.), 10% fetal bovine serum, and purified Trx-OBM (0-100 ng/ml)obtained in Example 15. The cell suspension was added to each well in48-well plates in the amount of 300 μl per well, and the cells werecultured at 37° C. for three days. After the culture broth was replacedwith the above-mentioned culture medium, the cells were cultured at 37°C. for four days. The cultured cells having tartaric acid resistant acidphosphatase activity (TRAP activity) were selectively stained accordingto the method described in Example 5. The number of stainedmultinucleates was measured by microscope observation. The results areshown in FIG. 29. It was confirmed that TRAP-positive multinucleateswere induced in a dose dependent manner by addition of Trx-OBM, while noTRAP-positive cells were detected in the wells to which Trx-OBM was notadded. Moreover, these TRAP-positive multinucleates were found positiveto vitronectin receptor which is a marker for osteoclasts. Furthermore,when similar cell culture was carried out on ivory slices placed on eachwell in a 48-well plate, pit formation was observed on the ivory slicesonly in the presence of Trx-OBM. Based on these findings, Trx-OBM wasformed to have the activity of inducing human osteoclast formation.

Example 40 Inhibition of Bone Resorbing Activity by Anti-OBM/sOBMAntibody

[⁴⁵Ca]—CaCl₂ solution (Amersham Co.) was subcutaneously injected into addY mouse (Japan SLC Co.) in the 15th day of pregnancy at a dose of 25μCi per mouse to label the bone of the fetus with ⁴⁵Ca. Next day, themouse was sacrificed to obtain the fetus. The forefoot of the fetus wasdrawn and the skin and muscle were removed to obtain the long bones. Thecartilage was removed to obtain the shafts of long bones. The shafts oflong bones were floated one by one in 0.5 ml of culture medium (BGJbmedium (GIBCO BRL company) containing a 0.2% bovine serum albumin (SigmaCo.)) in each well in 24-well plates, and cultured for 24 hours at 37°C. in 5% CO₂. After the pre-cultivation, the bones were transferred intovarious fresh culture media (0.5 ml), each containing one of differentbone resorbing factors (vitamin D₃, prostaglandins E₂, parathyroidhormone, interleukin lα.), and normal rabbit IgG (100 μg/ml; as acontrol), or the rabbit anti-OBM/sOBM polyclonal antibody prepared inExample 28, followed by further cultivation for 72 hours. After thecultivation, the long bones were placed in 0.5 ml of an aqueous solutionof 5% trichloroacetic acid (Wako Pure Chemicals Co., Ltd.), and allowedto stand at room temperature for more than 3 hours to decalcify. Five mlof a scintillator (AQUASOL-2, PACKARD Co.) was added to the culturebroth and the extract of the trichloroacetic acid solution (each 0.5 ml)to measure the radioactivity of ⁴⁵Ca, whereby the ratio of the ⁴⁵Cawhich was liberated into the culture broth by bone resorption wascalculated. The results are shown in FIGS. 30 to 33. As a result,vitamin D₃ (10⁻⁸ M) was found to increase the bone resorbing activity,but the rabbit anti-OBM/sOBM polyclonal antibody suppressed the boneresorption stimulated by vitamin D₃ in a concentration-dependent manner,completely inhibiting the increased bone resorption at a concentrationof 100 μg/ml (FIG. 30). Prostaglandins E₂ (10⁻⁶ M) and parathyroidhormone (100 ng/ml) also increased the bone resorbing activity. However,addition of 100 μg/ml of the rabbit anti-OBM/sOBM polyclonal antibodyalmost completely inhibited the bone resorption stimulated byprostaglandins E₂ and parathyroid hormone (FIGS. 31 and 32). On theother hand, normal rabbit IgG (100 μg/ml), which was used as a positivecontrol, did not affect the bone resorbing activity induced byprostaglandins E₂ and parathyroid hormone. Bone resorption was alsoincreased by interleukin 1α (10 μg/ml), but significantly inhibited bythe addition of rabbit anti-OBM/sOBM polyclonal antibody (100 μg/ml)(FIG. 33). Based on these results, it is clear that the antibody of thepresent invention is a superior substance as a bone resorptioninhibitor. The results obtained by similar experiment using H—OBM 9which is a mouse anti-human OBM/sOBM antibody, confirmed that thisantibody exhibits an almost equivalent bone resorption-inhibitory effectas the rabbit anti-OBM/sOBM polyclonal antibody.

INDUSTRIAL APPLICABILITY

The present invention provides a novel protein that specifically bindsto osteoclastogenesis-inhibitory factor (OCIF), a process for preparingthe protein, a screening method for a substance which controlsexpression of this protein using this protein, a screening method for asubstance which inhibits or modulates the activity of this protein, ascreening method for the receptor which transmits the activity of thisprotein by binding thereto, a pharmaceutical composition which containsthe substance obtained by these screening methods, an antibody for thesaid protein, and an agent for treating bone metabolism abnormalityusing the antibody.

Moreover, the present invention provides a DNA encoding a novel protein(OCIF-binding molecule) which binds to osteoclastogenesis-inhibit-oryfactor (OCIF), a protein which possesses an amino acid sequence encodedby the DNA, a method for preparing the protein specifically binding toOCIF using said DNA by a genetic engineering technique, and an agentcomprising said protein for treating bone metabolism acatastasia.Furthermore, the present invention provides a screening method for asubstance which controls expression of the OCIF-binding molecule, ascreening method for a substance which inhibits or modulates theactivity of the OCIF-binding molecule by binding thereto, a screeningmethod for the receptor which transmits the activity of the OCIF-bindingmolecule by binding thereto, and a pharmaceutical composition whichcontains the substance obtained by these screening methods.

Still further, the present invention provides a DNA encoding a novelhuman protein capable of binding to osteoclastogenesis-inhibitoryfactor, OCIF (human OCIF-binding molecule, human OBM), a proteincontaining an amino acid sequence encoded by the DNA, a process forpreparing a protein having characteristics of specifically binding toOCIF and exhibiting a biological activity to support and promote theosteoclast differentiation and maturation by means of geneticengineering technique, and an agent for treating bone metabolismabnormality using the protein. Furthermore, the present inventionprovides a screening method for a substance which controls expression ofthe OCIF-binding molecule, a screening method for a substance whichinhibits or modulates the activity of the OCIF-binding molecule bybinding thereto, a screening method for the receptor which transmits thebiological activity of the OCIF-binding molecule by binding thereto, apharmaceutical composition which contains the substance obtained bythese screening methods, an antibody to human OCIF-binding protein, andan agent for preventing and/or treating bone metabolism abnormalitysymptoms using the antibody.

In addition, the present invention provides antibodies which recognizeboth antigens (anti-OBM/sOBM antibodies), one is a membrane-boundprotein which specifically binds to OCIF (OCIF binding molecule; OBM)and the other a soluble OBM (sOBM) which does not have a membranebinding region, a process for preparing the antigen, a method formeasuring the OBM and sOBM using these antibodies, and an agent forpreventing and/or treating bone metabolism abnormality symptoms usingthe antibody as an effective component.

The protein and antibody prepared by the process of the presentinvention are useful as medicines and/or reagents for research and testpurposes.

DESCRIPTION OF DEPOSITED MICROORGANISMS

(1) Name and address of the depository organization to whichmicroorganism was deposited

Agency of Industrial Science and Technology

1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postal code 305)

Date of deposition to the depository organization

May 23, 1997

The deposition number

FERM BP-5953

(2) Name and address of the depository organization to whichmicroorganism was deposited

Agency of Industrial Science and Technology

1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postal code 305)

Date of deposition to the depository organization

Aug. 13, 1997

The deposition number

FERM BP-6058

(3) Name and address of the depository organization to whichmicroorganism was deposited

Agency of Industrial Science and Technology

1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postal code 305)

Date of deposition to the depository organization

Nov. 5, 1997 (Original deposition date)

The deposition number

FERM BP-6264

(4) Name and address of the depository organization to whichmicroorganism was deposited

Agency of Industrial Science and Technology

1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postal code 305)

Date of deposition to the depository organization

Nov. 5, 1997 (Original deposition date)

The deposition number

FERM BP-6265

(5) Name and address of the depository organization to whichmicroorganism was deposited

Agency of Industrial Science and Technology

1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan (postal code 305)

Date of deposition to the depository organization

Nov. 5, 1997 (Original deposition date)

The deposition number

FERM BP-6266

1. (canceled)
 2. A method for identifying a substance that modifies oneor more activities of OCIF-binding molecule (OBM), comprising:contacting cells expressing OBM with a test substance; and measuring anOBM-associated activity to identify whether the test substance modifiesone or more activities of OBM.
 3. The method of claim 2, wherein OBM isa human or mouse OBM.
 4. The method of claim 3, wherein OBM is asecreted-form OBM.
 5. The method of claim 2, wherein OBM comprises theamino acid sequence of SEQ ID NO: 11, SEQ ID NO: 17 or SEQ ID NO:
 19. 6.The method of claim 2, wherein the OBM-associated activity is osteoclastformation or bone resorption.
 7. The method of claim 2, wherein thecells comprise mononuclear leukocytes.
 8. The method of claim 2, whereinthe cells comprise osteoblastic stromal cells, preadipocyte cells,spleen cells, or a combination thereof.
 9. The method of claim 8,wherein the osteoblastic stromal cells are human or mouse osteoblasticstromal cells.
 10. The method of claim 9, wherein the mouse osteoblasticstromal cells are ST2 cells.
 11. The method of claim 2, wherein thecells are cultured in the presence of one or more osteotropic factors.12. The method of claim 11, wherein the one or more osteotropic factorsare selected from the group consisting of active-form vitamin D₃,parathyroid hormone (PTH), interleukin-1, interleukin-6, interleukin-11,Oncostatin M, and leukemia inhibitory factor.
 13. The method of claim11, wherein the one or more osteotropic factors are active-form vitaminD₃ or PTH.
 14. The method of claim 2, wherein the measuring of theOBM-associated activity comprises detecting tartaric acid resistant acidphosphatase (TRAP) activity.
 15. The method of claim 2, wherein themeasuring of the OBM-associated activity comprises detecting thepresence of vitronectin receptors.
 16. The method of claim 2, whereinthe test substance inhibits one or more activities of the OBM.
 17. Themethod of claim 16, wherein the inhibition of OBM by the test substanceis identified by decreased osteoclast formation in the presence of thetest substance as compared to the absence of the test substance.
 18. Themethod of claim 2, wherein the test substance increases one or moreactivities of the OBM.
 19. The method of claim 18, wherein the increasedactivity of OBM by the test substance is identified by increasedosteoclast formation in the presence of the test substance as comparedto the absence of the test substance.
 20. The method of claim 2, furthercomprising determining whether said substance binds to OBM.
 21. A methodfor identifying a modulator of OCIF-binding molecule (OBM), comprising:contacting an OBM with one or more test substances; and measuring thespecific binding between the one or more test substances and the OBM toidentify whether the test substance is a modulator of OBM.
 22. Themethod of claim 21, wherein the method comprises screening a peptidelibrary.